Borrelia burgdorferi locus BB0795 encodes a BamA orthologue required for growth and efficient localization of outer membrane proteins

The outer membrane (OM) of the pathogenic diderm spirochete, Borrelia burgdorferi, contains integral β‐barrel outer membrane proteins (OMPs) in addition to its numerous outer surface lipoproteins. Very few OMPs have been identified in B. burgdorferi, and the protein machinery required for OMP assembly and OM localization is currently unknown. Essential OM BamA proteins have recently been characterized in Gram‐negative bacteria that are central components of an OM β‐barrel assembly machine and are required for proper localization and insertion of bacterial OMPs. In the present study, we characterized a putative B. burgdorferi BamA orthologue encoded by open reading frame bb0795. Structural model predictions and cellular localization data indicate that the B. burgdorferi BB0795 protein contains an N‐terminal periplasmic domain and a C‐terminal, surface‐exposed β‐barrel domain. Additionally, assays with an IPTG‐regulatable bb0795 mutant revealed that BB0795 is required for B. burgdorferi growth. Furthermore, depletion of BB0795 results in decreased amounts of detectable OMPs in the B. burgdorferi OM. Interestingly, a decrease in the levels of surface‐exposed lipoproteins was also observed in the mutant OMs. Collectively, our structural, cellular localization and functional data are consistent with the characteristics of other BamA proteins, indicating that BB0795 is a B. burgdorferi BamA orthologue.     

Host cell heparan sulfate glycosaminoglycans are ligands for OspF‐related proteins of the Lyme disease spirochete

Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissues such as heart, joints and the nervous tissues. Host glycosaminoglycans, highly modified repeating disaccharides that are present on cell surfaces and in extracellular matrix, are common targets of microbial pathogens during tissue colonization. While several dermatan sulfate‐binding B. burgdorferi adhesins have been identified, B. burgdorferi adhesins documented to promote spirochetal binding to heparan sulfate have not yet been identified. OspEF‐related proteins (Erps), a large family of plasmid‐encoded surface lipoproteins that are produced in the mammalian host, can be divided into the OspF‐related, OspEF‐leader peptide (Elp) and OspE‐related subfamilies. We show here that a member of the OspF‐related subfamily, ErpG, binds to heparan sulfate and when produced on the surface of an otherwise non‐adherent B. burgdorferi strain, ErpG promotes heparan sulfate‐mediated bacterial attachment to the glial but not the endothelial, synovial or respiratory epithelial cells. Six other OspF‐related proteins were capable of binding heparan sulfate, whereas representative OspE‐related and Elp proteins lacked this activity. These results indicate that OspF‐related proteins are heparan sulfate‐binding adhesins, at least one of which promotes bacterial attachment to glial cells.     

Crystal stuctures of MglB‐2 (TP0684), a topologically variant d‐glucose‐binding protein from Treponema pallidum,reveal a ligand‐induced conformational change

Previously, we determined the crystal structure of apo‐TpMglB‐2, a d ‐glucose‐binding component of a putative ABC transporter from the syphilis spirochete Treponema pallidum. The protein had an unusual topology for this class of proteins, raising the question of whether the d ‐glucose‐binding mode would be different in TpMglB‐2. Here, we present the crystal structures of a variant of TpMglB‐2 with and without d ‐glucose bound. The structures demonstrate that, despite its aberrant topology, the protein undergoes conformational changes and binds d ‐glucose similarly to other Mgl‐type proteins, likely facilitating d ‐glucose uptake in T. pallidum.     

Detection of Lyme disease spirochete,Borrelia burgdorferi sensu lato,including three novel genotypes in ticks (Acari: Ixodidae) collected from songbirds (Passeriformes) across Canada

Lyme disease is reported across Canada, but pinpointing the source of infection has been problematic. In this three‐year, bird‐tick‐pathogen study (2004–2006), 366 ticks representing 12 species were collected from 151 songbirds (31 passerine species/subspecies) at 16 locations Canada‐wide. Of the 167 ticks/pools tested, 19 (11.4%) were infected with Borrelia burgdorferi sensu lato (s.l.). Sequencing of the rrf‐rrl intergenic spacer gene revealed four Borrelia genotypes: B. burgdorferi sensu stricto (s.s.) and three novel genotypes (BC genotype 1, BC genotype 2, BC genotype 3). All four genotypes were detected in spirochete‐infected Ixodes auritulus (females, nymphs, larvae) suggesting this tick species is a vector for B. burgdorferi s.l. We provide first‐time records for: ticks in the Yukon (north of 60° latitude), northernmost collection of Amblyomma americanum in North America, and Amblyomma imitator in Canada. First reports of bird‐derived ticks infected with B. burgdorferi s.l. include: live culture of spirochetes from Ixodes pacificus (nymph) plus detection in I. auritulus nymphs, Ixodes scapularis in New Brunswick, and an I. scapularis larva in Canada. We provide the first account of B. burgdorferi s. l. in an Ixodes muris tick collected from a songbird anywhere. Congruent with previous data for the American Robin, we suggest that the Common Yellowthroat, Golden‐crowned Sparrow, Song Sparrow, and Swainson's Thrush are reservoir‐competent hosts. Song Sparrows, the predominant hosts, were parasitized by I. auritulus harboring all four Borrelia genotypes. Our results show that songbirds import B. burgdorferi s.l.‐infected ticks into Canada. Bird‐feeding I. scapularis subadults were infected with Lyme spirochetes during both spring and fall migration in eastern Canada. Because songbirds disperse millions of infected ticks across Canada, people and domestic animals contract Lyme disease outside of the known and expected range.     

The importance of passerine birds as tick hosts and in the transmission of Borrelia burgdorferi,the agent of Lyme disease: a case study from Scotland

Borrelia burgdorferi sensu lato (s.l.) is the causative agent of Lyme borreliosis, the most common tick‐borne zoonosis of humans in Europe and North America. Here, we assessed the relative importance of different passerine bird species as tick hosts and their contribution to the B. burgdorferi s.l. transmission cycle in a rural residential area in Scotland. We caught 1229 birds of 22 species during the tick‐questing season. On average, 29% carried larval ticks (0.8 larvae per individual) and 5% carried nymph ticks (0.06 nymphs per individual). All attached ticks tested were Ixodes ricinus. Using a nested‐PCR, we found that 20% of nymphs tested positive to B. burgdorferi s.l. and all these were of the genospecies Borrelia garinii. We identified two new bird species carrying infected nymphs: Eurasian Siskin Carduelis spinus and European Greenfinch Carduelis chloris. Ground‐foraging species were more important than arboreal species in hosting I. ricinus nymphs and B. burgdorferi s.l. Common Blackbirds Turdus merula were the most common hosts, with Song Thrushes Turdus philomelos, Dunnocks Prunella modularis, European Greenfinches and Chaffinches Fringilla coelebs also hosting high rates of infection.     

Analysis of a Borrelia burgdorferi phosphodiesterase demonstrates a role for cyclic‐di‐guanosine monophosphate in motility and virulence

The genome of Borrelia burgdorferi encodes a set of genes putatively involved in cyclic‐dimeric guanosine monophosphate (cyclic‐di‐GMP) metabolism. Although BB0419 was shown to be a diguanylate cyclase, the extent to which bb0419 or any of the putative cyclic‐di‐GMP metabolizing genes impact B. burgdorferi motility and pathogenesis has not yet been reported. Here we identify and characterize a phosphodiesterase (BB0363). BB0363 specifically hydrolyzed cyclic‐di‐GMP with a K_m of 0.054 µM, confirming it is a functional cyclic‐di‐GMP phosphodiesterase. A targeted mutation in bb0363 was constructed using a newly developed promoterless antibiotic cassette that does not affect downstream gene expression. The mutant cells exhibited an altered swimming pattern, indicating a function for cyclic‐di‐GMP in regulating B. burgdorferi motility. Furthermore, the bb0363 mutant cells were not infectious in mice, demonstrating an important role for cyclic‐di‐GMP in B. burgdorferi infection. The mutant cells were able to survive within Ixodes scapularis ticks after a blood meal from naïve mice; however, ticks infected with the mutant cells were not able to infect naïve mice. Both motility and infection phenotypes were restored upon genetic complementation. These results reveal an important connection between cyclic‐di‐GMP, B. burgdorferi motility and Lyme disease pathogenesis. A mechanism by which cyclic‐di‐GMP influences motility and infection is proposed.     

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol‐3‐phosphate‐binding proteins

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol‐3‐phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P‐binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P‐binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P‐binding site, or by a secreted PI4P‐binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P‐binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P‐binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.     

Molecular cloning,expression profile,odorant affinity,and stability of two odorant‐binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant‐binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant‐binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real‐time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant‐binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans‐caryophelle, farnesene, and cis‐3‐Hexen‐1‐ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild‐type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α‐helical content.     

Characterization of 6S RNA in the Lyme disease spirochete

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity.     

Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi

Hfq is a global regulatory RNA‐binding protein. We have identified and characterized an atypical Hfq required for gene regulation and infectivity in the Lyme disease spirochete Borrelia burgdorferi. Sequence analyses of the putative B. burgdorferi Hfq protein revealed only a modest level of similarity with the Hfq from Escherichia coli, although a few key residues are retained and the predicted tertiary structure is similar. Several lines of evidence suggest that the B. burgdorferi bb0268 gene encodes a functional Hfq homologue. First, the hfq_Bb gene (bb0268) restores the efficient translation of an rpoS::lacZ fusion in an E. coli hfq null mutant. Second, the Hfq from B. burgdorferi binds to the small RNA DsrA_Bb and the rpoS mRNA. Third, a B. burgdorferi hfq null mutant was generated and has a pleiotropic phenotype that includes increased cell length and decreased growth rate, as found in hfq mutants in other bacteria. The hfq_Bb mutant phenotype is complemented in trans with the hfq gene from either B. burgdorferi or, surprisingly, E. coli. This is the first example of a heterologous bacterial gene complementing a B. burgdorferi mutant. The alternative sigma factor RpoS and the outer membrane lipoprotein OspC, which are induced by increased temperature and required for mammalian infection, are not upregulated in the hfq mutant. Consequently, the hfq mutant is not infectious by needle inoculation in the murine model. These data suggest that Hfq plays a key role in the regulation of pathogenicity factors in B. burgdorferi and we hypothesize that the spirochete has a complex Hfq‐dependent sRNA network.     

UNCOORDINATED PHYLOGEOGRAPHY OF BORRELIA BURGDORFERI AND ITS TICK VECTOR,IXODES SCAPULARIS

Vector‐borne microbes necessarily co‐occur with their hosts and vectors, but the degree to which they share common evolutionary or biogeographic histories remains unexplored. We examine the congruity of the evolutionary and biogeographic histories of the bacterium and vector of the Lyme disease system, the most prevalent vector‐borne disease in North America. In the eastern and midwestern US, Ixodes scapularis ticks are the primary vectors of Borrelia burgdorferi, the bacterium that causes Lyme disease. Our phylogeographic and demographic analyses of the 16S mitochondrial rDNA suggest that northern I. scapularis populations originated from very few migrants from the southeastern US that expanded rapidly in the Northeast and subsequently in the Midwest after the recession of the Pleistocene ice sheets. Despite this historical gene flow, current tick migration is restricted even between proximal sites within regions. In contrast, B. burgdorferi suffers no barriers to gene flow within the northeastern and midwestern regions but shows clear interregional migration barriers. Despite the intimate association of B. burgdorferi and I. scapularis, the population structure, evolutionary history, and historical biogeography of the pathogen are all contrary to its arthropod vector. In the case of Lyme disease, movements of infected vertebrate hosts may play a larger role in the contemporary expansion and homogenization of the pathogen than the movement of tick vectors whose populations continue to bear the historical signature of climate‐induced range shifts.     

Effects of a zoonotic pathogen,Borrelia burgdorferi,on the behavior of a key reservoir host

Most emerging infectious diseases of humans are transmitted to humans from other animals. The transmission of these “zoonotic” pathogens is affected by the abundance and behavior of their wildlife hosts. However, the effects of infection with zoonotic pathogens on behavior of wildlife hosts, particularly those that might propagate through ecological communities, are not well understood. Borrelia burgdorferi is a bacterium that causes Lyme disease, the most common vector‐borne disease in the USA and Europe. In its North American range, the pathogen is most frequently transmitted among hosts through the bite of infected blacklegged ticks (Ixodes scapularis). Using sham and true vaccines, we experimentally manipulated infection load with this zoonotic pathogen in its most competent wildlife reservoir host, the white‐footed mouse, Peromyscus leucopus, and quantified the effects of infection on mouse foraging behavior, as well as levels of mouse infestation with ticks. Mice treated with the true vaccine had 20% fewer larval blacklegged ticks infesting them compared to mice treated with the sham vaccine, a significant difference. We observed a nonsignificant trend for mice treated with the true vaccine to be more likely to visit experimental foraging trays (20%–30% effect size) and to prey on gypsy moth pupae (5%–20% effect size) compared to mice treated with the sham vaccine. We observed no difference between mice on true‐ versus sham‐vaccinated grids in risk‐averse foraging. Infection with this zoonotic pathogen appears to elicit behavioral changes that might reduce self‐grooming, but other behaviors were affected subtly or not at all. High titers of B. burgdorferi in mice could elicit a self‐reinforcing feedback loop in which reduced grooming increases tick burdens and hence exposure to tick‐borne pathogens.     

Interaction of the Lyme disease spirochete with its tick vector

Borrelia burgdorferi, the causative agent of Lyme disease (along with closely related genospecies), is in the deeply branching spirochete phylum. The bacterium is maintained in nature in an enzootic cycle that involves transmission from a tick vector to a vertebrate host and acquisition from a vertebrate host to a tick vector. During its arthropod sojourn, B. burgdorferi faces a variety of stresses, including nutrient deprivation. Here, we review some of the spirochetal factors that promote persistence, maintenance and dissemination of B. burgdorferi in the tick, and then focus on the utilization of available carbohydrates as well as the exquisite regulatory systems invoked to adapt to the austere environment between blood meals and to signal species transitions as the bacteria traverse their enzootic cycle. The spirochetes shift their source of carbon and energy from glucose in the vertebrate to glycerol in the tick. Regulation of survival under limiting nutrients requires the classic stringent response in which Rel_Bbu controls the levels of the alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively termed (p)ppGpp), while regulation at the tick–vertebrate interface as well as regulation of protective responses to the blood meal require the two‐component system Hk1/Rrp1 to activate production of the second messenger cyclic‐dimeric‐GMP (c‐di‐GMP).