Cytoarchitecture of ex vivo midgut cultures of unfed Ixodes scapularis infected with a tick-borne flavivirus.

A bite from an infected tick is the primary means of transmission for tick-borne flaviviruses (TBFV). Ticks ingest the virus while feeding on infected blood. The traditional view is that the virus first replicates in and transits the tick midgut prior to dissemination to other organs, including salivary glands. Thus, understanding TBFV infection in the tick midgut is a key first step in identifying potential countermeasures against infection. Ex vivo midgut cultures prepared from unfed adult female Ixodes scapularis ticks were viable and remained morphologically intact for more than 8 days. The midgut consisted of two clearly defined cell layers separated by a basement membrane: an exterior network of smooth muscle cells and an internal epithelium composed of digestive generative cells. The smooth muscle cells were arranged in a stellate circumferential pattern spaced at regular intervals along the long axis of midgut diverticula. When the cultures were infected with the TBFV Langat virus (LGTV), virus production increased by two logs with a peak at 96 hours post-infection. Infected cells were readily identified by immunofluorescence staining for the viral envelope protein, nonstructural protein 3 (NS3) and dsRNA. Microscopy of the stained cultures suggested that generative cells were the primary target for virus infection in the midgut. Infected cells exhibited an expansion of membranes derived from the endoplasmic reticulum; a finding consistent with TBFV infected cell cultures. Electron microscopy of infected cultures revealed virus particles in the basolateral region between epithelial cells. These results demonstrated LGTV replication in midgut generative cells of artificially infected, ex vivo cultures of unfed adult female I. scapularis ticks.

Of Murines and Humans: Modeling Persistent Powassan Disease in C57BL/6 Mice.

Powassan infection is caused by two closely related, tick-transmitted viruses of the genus Flavivirus (family Flaviviridae): Powassan virus lineage I (POWV) and lineage II (known as deer tick virus [DTV]). Infection is typically asymptomatic or mild but can progress to neuroinvasive disease. Approximately 10% of neuroinvasive cases are fatal, and half of the survivors experience long-term neurological sequelae. Understanding how these viruses cause long-term symptoms as well as the possible role of viral persistence is important for developing therapies. We intraperitoneally inoculated 6-week-old C57BL/6 mice (50% female) with 103 focus-forming units (FFU) DTV and assayed for infectious virus, viral RNA, and inflammation during acute infection and 21, 56, and 84 days postinfection (dpi). Although most mice (86%) were viremic 3 dpi, only 21% of the mice were symptomatic and 83% recovered. Infectious virus was detected only in the brains of mice sampled during the acute infection. Viral RNA was detected in the brain until 84 dpi, but the magnitude decreased over time. Meningitis and encephalitis were visible in acute mice and from mice sampled at 21 dpi. Inflammation was observed until 56 dpi in the brain and 84 dpi in the spinal cord, albeit at low levels. These results suggest that the long-term neurological symptoms associated with Powassan disease are likely caused by lingering viral RNA and chronic inflammation in the central nervous system rather than by a persistent, active viral infection. The C57BL/6 model of persistent Powassan mimics illness in humans and can be used to study the mechanisms of chronic disease. IMPORTANCE Half of Powassan infection survivors experience long-term, mild to severe neurological symptoms. The progression from acute to chronic Powassan disease is not well understood, severely limiting treatment and prevention options. Infection of C57BL/6 mice with DTV mimics clinical disease in humans, and the mice exhibit CNS inflammation and viral RNA persistence until at least 86 dpi, while infectious virus is undetectable after 12 dpi. These findings suggest that the long-term neurological symptoms of chronic Powassan disease are in part due the persistence of viral RNA and the corresponding long-term inflammation of the brain and spinal cord. Our work demonstrates that C57BL/6 mice can be used to study the pathogenesis of chronic Powassan disease.

Kinetics of tick infection by the relapsing fever spirochete Borrelia hermsii acquired through artificial membrane feeding chambers.

The relapsing fever agent Borrelia hermsii is transmitted by the tick Ornithodoros hermsi. To study the B. hermsii-tick interactions required for pathogen acquisition and transmission we developed an artificial membrane feeding system for O. hermsi nymphs and adults that results in a high percentage of engorgement. This system provides the nutritional requirements necessary for the tick to develop, mate, and produce viable eggs. By inoculating the blood with B. hermsii, we were able to obtain infected ticks for quantitative studies on pathogen acquisition and persistence. These ticks subsequently transmitted the spirochetes to mice, validating this system for both acquisition and transmission studies. Using this feeding method, a mutant of the antigenic variation locus of B. hermsii (Vmp-) that is incapable of persisting in mice was acquired by ticks at equivalent densities as the wild-type. Furthermore, Vmp is not required for persistence in the tick, as the mutant and wild-type strains are maintained at similar numbers after ecdysis and subsequent feeding. These results support the theory that Vmp is an adaptation for mammalian infection but unnecessary for survival within the tick. Interestingly, B. hermsii numbers severely declined after acquisition, though these ticks still transmitted the infection to mice. This procedure reduces animal use and provides a safe, highly controlled and well-contained alternative method for feeding and maintaining O. hermsi colonies. Importantly, this system permits quantitative studies with B. hermsii strains through ingestion during the blood meal, and thus more closely recapitulates pathogen acquisition in nature than other artificial systems.

Microbiomes of Blood-Feeding Arthropods: Genes Coding for Essential Nutrients and Relation to Vector Fitness and Pathogenic Infections. A Review.

BACKGROUND: Blood-feeding arthropods support a diverse array of symbiotic microbes, some of which facilitate host growth and development whereas others are detrimental to vector-borne pathogens. We found a common core constituency among the microbiota of 16 different arthropod blood-sucking disease vectors, including Bacillaceae, Rickettsiaceae, Anaplasmataceae, Sphingomonadaceae, Enterobacteriaceae, Pseudomonadaceae, Moraxellaceae and Staphylococcaceae. By comparing 21 genomes of common bacterial symbionts in blood-feeding vectors versus non-blooding insects, we found that certain enteric bacteria benefit their hosts by upregulating numerous genes coding for essential nutrients. Bacteria of blood-sucking vectors expressed significantly more genes (p < 0.001) coding for these essential nutrients than those of non-blooding insects. Moreover, compared to endosymbionts, the genomes of enteric bacteria also contained significantly more genes (p < 0.001) that code for the synthesis of essential amino acids and proteins that detoxify reactive oxygen species. In contrast, microbes in non-blood-feeding insects expressed few gene families coding for these nutrient categories. We also discuss specific midgut bacteria essential for the normal development of pathogens (e.g., Leishmania) versus others that were detrimental (e.g., bacterial toxins in mosquitoes lethal to Plasmodium spp.).

Probing the Role of bba30, a Highly Conserved Gene of the Lyme Disease Spirochete, Throughout the Mouse-Tick Infectious Cycle.

Borrelia burgdorferi, the causative agent of Lyme disease, has a complex and segmented genome consisting of a small linear chromosome and up to 21 linear and circular plasmids. Some of these plasmids are essential as they carry genes that are critical during the life cycle of the Lyme disease spirochete. Among these is a highly conserved linear plasmid, lp54, which is crucial for the mouse-tick infectious cycle of B. burgdorferi. However, the functions of most lp54-encoded open reading frames (ORFs) remain unknown. In this study, we investigate the contribution of a previously uncharacterized lp54 gene during the infectious cycle of B. burgdorferi. This gene, bba30, is conserved in the Borrelia genus but lacks any identified homologs outside the genus. Homology modeling of BBA30 ORF indicated the presence of a nucleic acid binding motif, helix-turn-helix (HTH), near the amino terminus of the protein, suggesting a putative regulatory function. A previous study reported that spirochetes with a transposon insertion in bba30 exhibited a noninfectious phenotype in mice. In the current study, however, we demonstrate that the highly conserved bba30 gene is not required by the Lyme disease spirochete at any stage of the experimental mouse-tick infectious cycle. We conclude that the undefined circumstances under which bba30 potentially confers a fitness advantage in the natural life cycle of B. burgdorferi are not factors of the experimental infectious cycle that we employ.

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions.

The deer tick Ixodes scapularis transmits a variety of disease agents in the United States, spreading the bacteria that causes Lyme borreliosis, the protozoan agent of babesiosis, and viruses such as Powassan. However, a variety of other organisms have also evolved symbiotic relationships with this tick species, and it seems likely that some of these microbes have simultaneously coevolved mechanisms to impact each other and their tick host. The number of organisms identified as I. scapularis symbionts has increased seemingly exponentially with the advent of PCR and next generation sequencing technologies, but convincing arguments have proposed that some of these are of environmental origin, unadapted to surviving the physiological conditions of the tick or that they are artifacts of ultrasensitive detection methods. In this review, we examine the diversity of the known microbes occurring within the I. scapularis microbiome, the evidence for interactions between microbes, and discuss whether some organisms reported to be symbionts of I. scapularis are experimental artifacts.

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes.

Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.-attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

The Lyme disease spirochete's BpuR DNA/RNA-binding protein is differentially expressed during the mammal-tick infectious cycle, which affects translation of the SodA superoxide dismutase.

When the Lyme disease spirochete, Borrelia burgdorferi, transfers from a feeding tick into a human or other vertebrate host, the bacterium produces vertebrate-specific proteins and represses factors needed for arthropod colonization. Previous studies determined that the B. burgdorferi BpuR protein binds to its own mRNA and autoregulates its translation, and also serves as co-repressor of erp transcription. Here, we demonstrate that B. burgdorferi controls transcription of bpuR, expressing high levels of bpuR during tick colonization but significantly less during mammalian infection. The master regulator of chromosomal replication, DnaA, was found to bind specifically to a DNA sequence that overlaps the bpuR promoter. Cultured B. burgdorferi that were genetically manipulated to produce elevated levels of BpuR exhibited altered levels of several proteins, although BpuR did not impact mRNA levels. Among these was the SodA superoxide dismutase, which is essential for mammalian infection. BpuR bound to sodA mRNA in live B. burgdorferi, and a specific BpuR-binding site was mapped 5' of the sodA open reading frame. Recognition of posttranscriptional regulation of protein levels by BpuR adds another layer to our understanding of the B. burgdorferi regulome, and provides further evidence that bacterial protein levels do not always correlate directly with mRNA levels.

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to basidiomycete transcripts dominated, and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate-active enzymes (CAZymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases, including highly expressed cellulases or hemicellulases. The CAZymes also included 162 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies.

Borrelia burgdorferi SpoVG DNA- and RNA-Binding Protein Modulates the Physiology of the Lyme Disease Spirochete.

The SpoVG protein of Borrelia burgdorferi, the Lyme disease spirochete, binds to specific sites of DNA and RNA. The bacterium regulates transcription of spoVG during the natural tick-mammal infectious cycle and in response to some changes in culture conditions. Bacterial levels of spoVG mRNA and SpoVG protein did not necessarily correlate, suggesting that posttranscriptional mechanisms also control protein levels. Consistent with this, SpoVG binds to its own mRNA, adjacent to the ribosome-binding site. SpoVG also binds to two DNA sites in the glpFKD operon and to two RNA sites in glpFKD mRNA; that operon encodes genes necessary for glycerol catabolism and is important for colonization in ticks. In addition, spirochetes engineered to dysregulate spoVG exhibited physiological alterations.IMPORTANCEB. burgdorferi persists in nature by cycling between ticks and vertebrates. Little is known about how the bacterium senses and adapts to each niche of the cycle. The present studies indicate that B. burgdorferi controls production of SpoVG and that this protein binds to specific sites of DNA and RNA in the genome and transcriptome, respectively. Altered expression of spoVG exerts effects on bacterial replication and other aspects of the spirochete's physiology.

A widely conserved bacterial cytoskeletal component influences unique helical shape and motility of the spirochete Leptospira biflexa.

Leptospires and other members of the evolutionarily ancient phylum of Spirochaetes are bacteria often characterized by long, highly motile spiral- or wave-shaped cells. Morphology and motility are critical factors in spirochete physiology, contributing to the ability of these bacteria to successfully colonize diverse environments. However, the mechanisms conferring the helical structure of Leptospira spp. have yet to be fully elucidated. We have identified five Leptospira biflexa bactofilin proteins, a recently characterized protein family with cytoskeletal properties. These five bactofilins are conserved in all species of the Leptospiraceae, indicating that these proteins arose early in the evolution of this family. One member of this protein family, LbbD, confers the optimal pitch distance in the helical structure of L. biflexa. Mutants lacking lbbD display a unique compressed helical morphology, a reduced motility and a decreased ability to tolerate cell wall stressors. The change in the helical spacing, combined with the motility and cell wall integrity defects, showcases the intimate relationship and coevolution between shape and motility in these spirochetes.

Physiologic and Genetic Factors Influencing the Zoonotic Cycle of Borrelia burgdorferi.

Borrelia burgdorferi is a symbiont of ticks of the Ixodes ricinus complex. These ticks serve as vectors to disseminate the spirochete to a variety of susceptible vertebrate hosts, which, in turn, act as reservoirs for naïve ticks to become infected, perpetuating the infectious life cycle of B. burgdorferi. The pivotal role of ticks in this life cycle and tick-spirochete interactions are the focus of this chapter. Here, we describe the challenging physiological environment that spirochetes encounter within Ixodes ticks, and the genetic factors that B. burgdorferi uses to successfully infect, persist, and be transmitted from the vector.

Function of the Borrelia burgdorferi FtsH Homolog Is Essential for Viability both In Vitro and In Vivo and Independent of HflK/C.

UNLABELLED: In many bacteria, the FtsH protease and its modulators, HflK and HflC, form a large protein complex that contributes to both membrane protein quality control and regulation of the cellular response to environmental stress. Both activities are crucial to the Lyme disease pathogen Borrelia burgdorferi, which depends on membrane functions, such as motility, protein transport, and cell signaling, to respond to rapid changes in its environment. Using an inducible system, we demonstrate that FtsH production is essential for both mouse and tick infectivity and for in vitro growth of B. burgdorferi FtsH depletion in B. burgdorferi cells resulted in membrane deformation and cell death. Overproduction of the protease did not have any detectable adverse effects on B. burgdorferi growth in vitro, suggesting that excess FtsH does not proteolytically overwhelm its substrates. In contrast, we did not observe any phenotype for cells lacking the protease modulators HflK and HflC (ΔHflK/C), although we examined morphology, growth rate, growth under stress conditions, and the complete mouse-tick infectious cycle. Our results demonstrate that FtsH provides an essential function in the life cycle of the obligate pathogen B. burgdorferi but that HflK and HflC do not detectably affect FtsH function. IMPORTANCE: Lyme disease is caused by Borrelia burgdorferi, which is maintained in nature in an infectious cycle alternating between small mammals and Ixodes ticks. B. burgdorferi produces specific membrane proteins to successfully infect and persist in these diverse organisms. We hypothesized that B. burgdorferi has a specific mechanism to ensure that membrane proteins are properly folded and biologically active when needed and removed if improperly folded or dysfunctional. Our experiments demonstrate that FtsH, a protease that fulfills this role in other microorganisms, is essential to B. burgdorferi viability. Cells depleted of FtsH do not survive in laboratory culture medium and cannot colonize mice or ticks, revealing an absolute requirement for this protease. However, the loss of two potential modulators of FtsH activity, HflK and HflC, does not detectably affect B. burgdorferi physiology. Our results provide the groundwork for the identification of FtsH substrates that are critical for the bacterium's viability.

Transcriptome and Secretome Analyses of the Wood Decay Fungus Wolfiporia cocos Support Alternative Mechanisms of Lignocellulose Conversion.

UNLABELLED: Certain wood decay basidiomycetes, collectively referred to as brown rot fungi, rapidly depolymerize cellulose while leaving behind the bulk of cell wall lignin as a modified residue. The mechanism(s) employed is unclear, but considerable evidence implicates the involvement of diffusible oxidants generated via Fenton-like chemistry. Toward a better understanding of this process, we have examined the transcriptome and secretome of Wolfiporia cocos when cultivated on media containing glucose, purified crystalline cellulose, aspen (Populus grandidentata), or lodgepole pine (Pinus contorta) as the sole carbon source. Compared to the results obtained with glucose, 30, 183, and 207 genes exhibited 4-fold increases in transcript levels in cellulose, aspen, and lodgepole pine, respectively. Mass spectrometry identified peptides corresponding to 64 glycoside hydrolase (GH) proteins, and of these, 17 corresponded to transcripts upregulated on one or both woody substrates. Most of these genes were broadly categorized as hemicellulases or chitinases. Consistent with an important role for hydroxyl radical in cellulose depolymerization, high transcript levels and upregulation were observed for genes involved in iron homeostasis, iron reduction, and extracellular peroxide generation. These patterns of regulation differ markedly from those of the closely related brown rot fungus Postia placenta and expand the number of enzymes potentially involved in the oxidative depolymerization of cellulose. IMPORTANCE: The decomposition of wood is an essential component of nutrient cycling in forest ecosystems. Few microbes have the capacity to efficiently degrade woody substrates, and the mechanism(s) is poorly understood. Toward a better understanding of these processes, we show that when grown on wood as a sole carbon source the brown rot fungus W. cocos expresses a unique repertoire of genes involved in oxidative and hydrolytic conversions of cell walls.

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species.

Motility is crucial for the infectious life cycle of Borrelia burgdorferi.

The Lyme disease spirochete, Borrelia burgdorferi, exists in a zoonotic cycle involving an arthropod tick and mammalian host. Dissemination of the organism within and between these hosts depends upon the spirochete's ability to traverse through complex tissues. Additionally, the spirochete outruns the host immune cells while migrating through the dermis, suggesting the importance of B. burgdorferi motility in evading host clearance. B. burgdorferi's periplasmic flagellar filaments are composed primarily of a major protein, FlaB, and minor protein, FlaA. By constructing a flaB mutant that is nonmotile, we investigated for the first time the absolute requirement for motility in the mouse-tick life cycle of B. burgdorferi. We found that whereas wild-type cells are motile and have a flat-wave morphology, mutant cells were nonmotile and rod shaped. These mutants were unable to establish infection in C3H/HeN mice via either needle injection or tick bite. In addition, these mutants had decreased viability in fed ticks. Our studies provide substantial evidence that the periplasmic flagella, and consequently motility, are critical not only for optimal survival in ticks but also for infection of the mammalian host by the arthropod tick vector.

Characterization of the Bat proteins in the oxidative stress response of Leptospira biflexa.

BACKGROUND: Leptospires lack many of the homologs for oxidative defense present in other bacteria, but do encode homologs of the Bacteriodes aerotolerance (Bat) proteins, which have been proposed to fulfill this function. Bat homologs have been identified in all families of the phylum Spirochaetes, yet a specific function for these proteins has not been experimentally demonstrated. RESULTS: We investigated the contribution of the Bat proteins in the model organism Leptospira biflexa for their potential contributions to growth rate, morphology and protection against oxidative challenges. A genetically engineered mutant strain in which all bat ORFs were deleted did not exhibit altered growth rate or morphology, relative to the wild-type strain. Nor could we demonstrate a protective role for the Bat proteins in coping with various oxidative stresses. Further, pre-exposing L. biflexa to sublethal levels of reactive oxygen species did not appear to induce a general oxidative stress response, in contrast to what has been shown in other bacterial species. Differential proteomic analysis of the wild-type and mutant strains detected changes in the abundance of a single protein only - HtpG, which is encoded by the gene immediately downstream of the bat loci. CONCLUSION: The data presented here do not support a protective role for the Leptospira Bat proteins in directly coping with oxidative stress as previously proposed. L. biflexa is relatively sensitive to reactive oxygen species such as superoxide and H2O2, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe.

Borrelia burgdorferi linear plasmid 38 is dispensable for completion of the mouse-tick infectious cycle.

Borrelia burgdorferi, the causative agent of Lyme disease, exists in a complex enzootic cycle, transiting between its vector, Ixodes ticks, and a diverse range of vertebrate hosts. B. burgdorferi linear plasmid 38 (lp38) contains several genes that are differentially regulated in response to conditions mimicking the tick or mouse environments, suggesting that these plasmid-borne genes may encode proteins important for the B. burgdorferi infectious cycle. Some of these genes encode potential virulence factors, including hypothetical lipoproteins as well as a putative membrane transport system. To characterize the role of lp38 in the B. burgdorferi infectious cycle, we constructed a shuttle vector to selectively displace lp38 from the B. burgdorferi genome and analyzed the resulting clones to confirm the loss of lp38. We found that, in vitro, clones lacking lp38 were similar to isogenic wild-type bacteria, both in growth rate and in antigenic protein production. We analyzed these strains in an experimental mouse-tick infectious cycle, and our results demonstrate that clones lacking lp38 are fully infectious in a mouse, can efficiently colonize the tick vector, and are readily transmitted to a naive host.

Use of the Cre-lox recombination system to investigate the lp54 gene requirement in the infectious cycle of Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, has a complex genome consisting of a linear chromosome and up to 21 linear and circular plasmids. These plasmids encode numerous proteins critical to the spirochete's infectious cycle and many hypothetical proteins whose functions and requirements are unknown. The conserved linear plasmid lp54 encodes several proteins important for survival in the mouse-tick infectious cycle, but the majority of the proteins are of unknown function and lack homologs outside the borreliae. In this study we adapted the Cre-lox recombination system to create large deletions in the B. burgdorferi genome. Using Cre-lox, we systematically investigated the contribution of 14 adjacent genes on the left arm of lp54 to the overall infectivity of B. burgdorferi. The deletion of the region of lp54 encompassing bba07 to bba14 had no significant effect on the infectious cycle of B. burgdorferi. The deletion of bba01 to bba07 resulted in a slight growth defect but did not significantly affect the ability of B. burgdorferi to complete the infectious cycle. This study demonstrated the utility of the Cre-lox system to efficiently explore gene requirements in B. burgdorferi and surprisingly revealed that a large number of the highly conserved proteins encoded on lp54 are not required to complete the infectious cycle.