A transwell assay method to evaluate Borrelia burgdorferi sensu stricto migratory chemoattraction toward tick saliva proteins.

We developed a transwell assay to quantify migration of the Lyme disease agent, Borrelia burgdorferi sensu stricto (s.s.), toward Ixodes scapularis salivary gland proteins. The assay was designed to assess B. burgdorferi s.s. migration upward against gravity through a transwell polycarbonate membrane overlaid with 6% gelatin. Borreliae that channeled into the upper transwell chamber in response to test proteins were enumerated by flow cytometry. The transwell assay measured chemoattractant activity for B. burgdorferi s.s. from salivary gland extract (SGE) harvested from nymphal ticks during bloodmeal engorgement on mice 42 h post-attachment and saliva collected from adult ticks. Additionally, SGE protein fractions separated by size exclusion chromatography demonstrated various levels of chemoattractant activity in the transwell assay. Sialostatin L, and Salp-like proteins 9 and 11 were identified by mass spectrometry in SGE fractions that exhibited elevated activity. Recombinant forms of these proteins were tested in the transwell assay and showed positive chemoattractant properties compared to controls and another tick protein, S15A. These results were reproducible providing evidence that the transwell assay is a useful method for continuing investigations to find tick saliva components instrumental in driving B. burgdorferi s.s. chemotaxis.

Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system.

Pathogens possess the ability to adapt and survive in some host species but not in others-an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations.

Lyme Disease in Humans.

Lyme disease (Lyme borreliosis) is a tick-borne, zoonosis of adults and children caused by genospecies of the Borrelia burgdorferi sensu lato complex. The ailment, widespread throughout the Northern Hemisphere, continues to increase globally due to multiple environmental factors, coupled with increased incursion of humans into habitats that harbor the spirochete. B. burgdorferi sensu lato is transmitted by ticks from the Ixodes ricinus complex. In North America, B. burgdorferi causes nearly all infections; in Europe, B. afzelii and B. garinii are most associated with human disease. The spirochete's unusual fragmented genome encodes a plethora of differentially expressed outer surface lipoproteins that play a seminal role in the bacterium's ability to sustain itself within its enzootic cycle and cause disease when transmitted to its incidental human host. Tissue damage and symptomatology (i.e., clinical manifestations) result from the inflammatory response elicited by the bacterium and its constituents. The deposition of spirochetes into human dermal tissue generates a local inflammatory response that manifests as erythema migrans (EM), the hallmark skin lesion. If treated appropriately and early, the prognosis is excellent. However, in untreated patients, the disease may present with a wide range of clinical manifestations, most commonly involving the central nervous system, joints, or heart. A small percentage (~10%) of patients may go on to develop a poorly defined fibromyalgia-like illness, post-treatment Lyme disease (PTLD) unresponsive to prolonged antimicrobial therapy. Below we integrate current knowledge regarding the ecologic, epidemiologic, microbiologic, and immunologic facets of Lyme disease into a conceptual framework that sheds light on the disorder that healthcare providers encounter.

The BB0345 Hypothetical Protein of Borrelia burgdorferi Is Essential for Mammalian Infection.

During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.

Comparison of growth and morphology of Borrelia burgdorferi sensu lato in BSK-H and BSK-II media stored for prolonged periods.

Barbour-Stoenner-Kelly II (BSK-II) and BSK-H media were used for cultivation and isolation of fastidious Borrelia species - the causative agents of Lyme borreliosis. Culture media have a limited shelf life and require adequate storage. Our goal was to assess how the growth of Borrelia would be affected by prolonged storage of media and inadequate storage conditions (BSK-H stored at +4 °C for 2.5 years and BSK-II stored at -20 °C for 11 years). Growth of different Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana strains was assessed during 2 weeks of incubation at 33 °C. Monitored parameters included cell count per mL, morphology and motility. The results of this study have shown weaker growth of borrelia strains in BSK-H at +4 °C (median final cell number of 1.5 × 106 /mL) than in BSK-II at -20 °C (median final cell number of 7.75 × 106 /mL) and in fresh BSK-H media (median final cell number of 8.95 × 106 /mL). Duration of storage of media had no impact on Borrelia morphology and motility. Our results indicate that temperature of -20 °C is optimal for long-term storage of medium, BSK-II stored for 11 years provided effective support to growth of Borrelia and may be employed for cultivation.

Cross-kingdom analysis of nymphal-stage Ixodes scapularis microbial communities in relation to Borrelia burgdorferi infection and load.

The tick microbiota may influence the colonization of Ixodes scapularis by Borrelia burgdorferi, the Lyme disease bacterium. Using conserved and pathogen-specific primers we performed a cross-kingdom analysis of bacterial, fungal, protistan and archaeal communities of I. scapularis nymphs (N = 105) collected from southern Vermont, USA. The bacterial community was dominated by a Rickettsia and several environmental taxa commonly reported in I. scapularis, as well as the human pathogens B. burgdorferi and Anaplasma phagocytophilum, agent of human granulocytic anaplasmosis. With the fungal primer set we detected primarily plant- and litter-associated taxa and >18% of sequences were Malassezia, a fungal genus associated with mammalian skin. Two 18S rRNA gene primer sets, intended to target protistan communities, returned mostly Ixodes DNA as well as the wildlife pathogen Babesia odocoilei (7% of samples), a Gregarines species (14%) and a Spirurida nematode (18%). Data from pathogen-specific and conserved primers were consistent in terms of prevalence and identification. We measured B. burgdorferi presence/absence and load and found that bacterial beta diversity varied based on B. burgdorferi presence/absence. Load was weakly associated with bacterial community composition. We identified taxa associated with B. burgdorferi infection that should be evaluated for their role in vector colonization by pathogens.

Genome-wide screen identifies novel genes required for Borrelia burgdorferi survival in its Ixodes tick vector.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction.

Detection of antibodies to Borrelia burgdorferi s.l. in wild small mammals and sensitivity of PCR and cultivation.

The aim of this study was to determine the seroprevalence of antibodies to B. burgdorferi s.l. in wild small mammals in the Czech Republic and compare sensitivity of PCR and cultivaton. Wild small mammals (n = 691) were trapped in years 2010-2014 in three localities of the Czech Republic. Heart rinses (n = 340) and sera (n = 351) were examined by modified indirect ELISA. Seventy animals were randomly selected for comparison of results of cultivation and PCR. Mean annual antiborelian positivity was 12% with statistical difference (p < 0.05) between Bank Vole (Clethrionomys glareolus) and other six animal species, while there was no statistical difference (p > 0.05) between rodentia and insectivora, gender and localities. The cultivation revealed one positive sample (1.4%), negative in both PCR and ELISA. Method PCR revealed seven positive samples (10%); two of them were simultaneously dubious in ELISA. Eleven animals, negative in cultivation and PCR, had antibodies in ELISA. Method of PCR compared to cultivation seems to be more sensitive for detection of Borrelia.

DksA Controls the Response of the Lyme Disease Spirochete Borrelia burgdorferi to Starvation.

The pathogenic spirochete Borrelia burgdorferi senses and responds to changes in the environment, including changes in nutrient availability, throughout its enzootic cycle in Ixodes ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response of B. burgdorferi to starvation. Wild-type and dksA mutant B. burgdorferi strains were subjected to starvation by shifting cultures grown in rich complete medium, Barbour-Stoenner-Kelly II (BSK II) medium, to a defined mammalian tissue culture medium, RPMI 1640, for 6 h under microaerobic conditions (5% CO2, 3% O2). Microarray analyses of wild-type B. burgdorferi revealed that genes encoding flagellar components, ribosomal proteins, and DNA replication machinery were downregulated in response to starvation. DksA mediated transcriptomic responses to starvation in B. burgdorferi, as the dksA-deficient strain differentially expressed only 47 genes in response to starvation compared to the 500 genes differentially expressed in wild-type strains. Consistent with a role for DksA in the starvation response of B. burgdorferi, fewer CFU of dksA mutants were observed after prolonged starvation in RPMI 1640 medium than CFU of wild-type B. burgdorferi spirochetes. Transcriptomic analyses revealed a partial overlap between the DksA regulon and the regulon of RelBbu, the guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase that controls the stringent response; the DksA regulon also included many plasmid-borne genes. Additionally, the dksA mutant exhibited constitutively elevated (p)ppGpp levels compared to those of the wild-type strain, implying a regulatory relationship between DksA and (p)ppGpp. Together, these data indicate that DksA, along with (p)ppGpp, directs the stringent response to effect B. burgdorferi adaptation to its environment.IMPORTANCE The Lyme disease bacterium Borrelia burgdorferi survives diverse environmental challenges as it cycles between its tick vectors and various vertebrate hosts. B. burgdorferi must withstand prolonged periods of starvation while it resides in unfed Ixodes ticks. In this study, the regulatory protein DksA is shown to play a pivotal role controlling the transcriptional responses of B. burgdorferi to starvation. The results suggest that DksA gene regulatory activity impacts B. burgdorferi metabolism, virulence gene expression, and the ability of this bacterium to complete its natural life cycle.

Investigating disease severity in an animal model of concurrent babesiosis and Lyme disease.

The incidence of babesiosis, Lyme disease and other tick-borne diseases has increased steadily in Europe and North America during the last five decades. Babesia microti is transmitted by species of Ixodes, the same ticks that transmit the Lyme disease-causing spirochete, Borrelia burgdorferi. B. microti can also be transmitted through transfusion of blood products and is the most common transfusion-transmitted infection in the U.S.A. Ixodes ticks are commonly infected with both B. microti and B. burgdorferi, and are competent vectors for transmitting them together into hosts. Few studies have examined the effects of coinfections on humans and they had somewhat contradictory results. One study linked coinfection with B. microti to a greater number of symptoms of overall disease in patients, while another report indicated that B. burgdorferi infection either did not affect babesiosis symptoms or decreased its severity. Mouse models of infection that manifest pathological effects similar to those observed in human babesiosis and Lyme disease offer a unique opportunity to thoroughly investigate the effects of coinfection on the host. Lyme disease has been studied using the susceptible C3H mouse infection model, which can also be used to examine B. microti infection to understand pathological mechanisms of human diseases, both during a single infection and during coinfections. We observed that high B. microti parasitaemia leads to low haemoglobin levels in infected mice, reflecting the anaemia observed in human babesiosis. Similar to humans, B. microti coinfection appears to enhance the severity of Lyme disease-like symptoms in mice. Coinfected mice have lower peak B. microti parasitaemia compared to mice infected with B. microti alone, which may reflect attenuation of babesiosis symptoms reported in some human coinfections. These findings suggest that B. burgdorferi coinfection attenuates parasite growth while B. microti presence exacerbates Lyme disease-like symptoms in mice.

N-acetylmannosamine (ManNAc) supports the growth of Borrelia burgdorferi in the absence of N-acetylglucosamine (GlcNAc).

Borrelia burgdorferi, the causative agent of Lyme disease, lacks the ability to biosynthesize many essential nutrients de novo, including N-acetylglucosamine (GlcNAc). This amino sugar is required for cell wall synthesis, and is a component of the complex growth medium used for in vitro propagation. When cultured without free GlcNAc, B. burgdorferi cells exhibit a unique biphasic growth pattern. We hypothesized that genes involved in the GlcNAc starvation response would be differentially expressed when compared to cells cultured in complete medium, and investigated this using transcriptomics. Twenty-one genes were differentially regulated in wild-type and starvation-adapted cells cultured without GlcNAc compared to wild-type cells cultured with GlcNAc. Of those, three genes involved in carbohydrate utilization were upregulated: bbb04 (chbC) encoding a subunit of the chitobiose transporter, bb0629 (fruA-2) encoding a putative carbohydrate transporter and bb0644 (nanE) encoding a putative GlcNAc-6-phosphate-2-epimerase predicted to catalyze the conversion of N-acetylmannosamine-6-phosphate (ManNAc-6-P) to GlcNAc-6-P. Quantitative RT-PCR was used to confirm differential expression of select genes, and substitution of free GlcNAc with free ManNAc resulted in growth to high cell density, suggesting B. burgdorferi cells can utilize free ManNAc for cell wall synthesis and energy production.

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.

Effect of Climate and Land Use on the Spatio-Temporal Variability of Tick-Borne Bacteria in Europe.

The incidence of tick-borne diseases caused by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. has been rising in Europe in recent decades. Early pre-assessment of acarological hazard still represents a complex challenge. The aim of this study was to model Ixodes ricinus questing nymph density and its infection rate with B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in five European countries (Italy, Germany, Czech Republic, Slovakia, Hungary) in various land cover types differing in use and anthropisation (agricultural, urban and natural) with climatic and environmental factors (Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), Land Surface Temperature (LST) and precipitation). We show that the relative abundance of questing nymphs was significantly associated with climatic conditions, such as higher values of NDVI recorded in the sampling period, while no differences were observed among land use categories. However, the density of infected nymphs (DIN) also depended on the pathogen considered and land use. These results contribute to a better understanding of the variation in acarological hazard for Ixodes ricinus transmitted pathogens in Central Europe and provide the basis for more focused ecological studies aimed at assessing the effect of land use in different sites on tick-host pathogens interaction.

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR.

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to humans through the bite of infected Ixodes ticks. The primary etiological agent in North America is Borrelia burgdorferi sensu stricto. As geographic risk regions expand, it is prudent to support robust surveillance programs that can measure tick infection rates, and communicate findings to clinicians, veterinarians, and the general public. The molecular technique of nested polymerase chain reaction (nPCR) has long been used for this purpose, and it remains a central, inexpensive, and robust approach in the detection of Borrelia in both ticks and wildlife. This article demonstrates the application of nPCR to tick DNA extracts to identify infected specimens. Two independent B. burgdorferi targets, genes encoding Flagellin B (FlaB) and Outer surface protein A (OspA), have been used extensively with this technique. The protocol involves tick collection, DNA extraction, and then an initial round of PCR to detect each of the two Borrelia-specific loci. Subsequent polymerase chain reaction (PCR) uses the product of the first reaction as a new template to generate smaller, internal amplification fragments. The nested approach improves upon both the specificity and sensitivity of conventional PCR. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.

Ketamine intervention limits pathogen expansion in vitro.

Ketamine is one of several clinically important drugs whose therapeutic efficacy is due in part to their ability to act upon ion channels prevalent in nearly all biological systems. In studying eukaryotic and prokaryotic organisms in vitro, we show that ketamine short-circuits the growth and spatial expansion of three microorganisms, Stachybotrys chartarum, Staphylococcus epidermidis and Borrelia burgdorferi, at doses efficient at reducing depression-like behaviors in mouse models of clinical depression. Although our findings do not reveal the mechanism(s) by which ketamine mediates its antifungal and antibacterial effects, we hypothesize that a function of L-glutamate signal transduction is associated with the ability of ketamine to limit pathogen expansion. In general, our findings illustrate the functional similarities between fungal, bacterial and human ion channels, and suggest that ketamine or its metabolites not only act in neurons, as previously thought, but also in microbial communities colonizing human body surfaces.

Peptide Uptake Is Essential for Borrelia burgdorferi Viability and Involves Structural and Regulatory Complexity of its Oligopeptide Transporter.

Borrelia burgdorferi is an extreme amino acid (AA) auxotroph whose genome encodes few free AA transporters and an elaborate oligopeptide transport system (B. burgdorferi Opp [BbOpp]). BbOpp consists of five oligopeptide-binding proteins (OBPs), two heterodimeric permeases, and a heterodimeric nucleotide-binding domain (NBD). Homology modeling based on the crystal structure of liganded BbOppA4 revealed that each OBP likely binds a distinct range of peptides. Transcriptional analyses demonstrated that the OBPs are differentially and independently regulated whereas the permeases and NBDs are constitutively expressed. A conditional NBD mutant failed to divide in the absence of inducer and replicated in an IPTG (isopropyl-ß-d-thiogalactopyranoside) concentration-dependent manner. NBD mutants grown without IPTG exhibited an elongated morphotype lacking division septa, often with flattening at the cell center due to the absence of flagellar filaments. Following cultivation in dialysis membrane chambers, NBD mutants recovered from rats not receiving IPTG also displayed an elongated morphotype. The NBD mutant was avirulent by needle inoculation, but infectivity was partially restored by oral administration of IPTG to infected mice. We conclude that peptides are a major source of AAs for B. burgdorferi both in vitro and in vivo and that peptide uptake is essential for regulation of morphogenesis, cell division, and virulence.IMPORTANCEBorrelia burgdorferi, the causative agent of Lyme disease, is an extreme amino acid (AA) auxotroph with a limited repertoire of annotated single-AA transporters. A major issue is how the spirochete meets its AA requirements as it transits between its arthropod vector and mammalian reservoir. While previous studies have confirmed that the B. burgdorferi oligopeptide transport (opp) system is capable of importing peptides, the importance of the system for viability and pathogenesis has not been established. Here, we evaluated the opp system structurally and transcriptionally to elucidate its ability to import a wide range of peptides during the spirochete's enzootic cycle. Additionally, using a novel mutagenesis strategy to abrogate opp transporter function, we demonstrated that peptide uptake is essential for bacterial viability, morphogenesis, and infectivity. Our studies revealed a novel link between borrelial physiology and virulence and suggest that peptide uptake serves an intracellular signaling function regulating morphogenesis and division.

Arthritis is inhibited in Borrelia-primed and infected interleukin-17A-deficient mice after administration of anti-gamma-interferon, anti-tumor necrosis factor alpha and anti-interleukin-6 antibodies.

The role that cytokines play in the induction of Lyme arthritis is gradually being delineated. We showed previously that severe arthritis developed in a T-cell-driven murine model, even in mice lacking interleukin-17A (IL-17A) and administered anti-gamma-interferon (IFN-γ) antibody. Increased levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), two pro-inflammatory cytokines, were detected in cultures of popliteal lymph node cells obtained from these mice. We hypothesized that concomitantly administered anti-IL-6, anti-TNF-α and anti-IFN-γ antibodies would inhibit the development of arthritis in IL-17A-deficient mice. Our results showed that swelling of the hind paws and histopathological changes consistent with arthritis were significantly reduced in IL-17A-deficient mice that administered the three anti-cytokine antibodies. These results suggest that treatment with multiple anti-cytokine antibodies can abrogate the induction of Lyme arthritis in mice.

Interactions between host immune response and antigenic variation that control Borrelia burgdorferi population dynamics.

The population dynamics of pathogens within hosts result from interactions between host immune responses and mechanisms of the pathogen to evade or resist immune responses. Vertebrate hosts have evolved adaptive immune responses to eliminate the infection, while many pathogens evade immune clearance through altering surface antigens. Such interactions can result in a characteristic pattern of pathogen population dynamics within hosts consisting of population growth after infection, rapid population decline following specific immune responses, followed by persistence at low densities during a chronic infection stage. Despite the medical importance of chronic infections, little is known about the conditions of the interactions between variable antigens and the adaptive immune system that cause the characteristic pathogen population dynamics. Using the vls antigenic variation system of the Lyme disease pathogen, Borrelia burgdorferi, as a model system, we investigated conditions of the interaction between the antigenic variation system and the adaptive immune response that can explain the within-host population dynamics of B. burgdorferi using mathematical modelling. This characteristic population dynamic pattern can be explained by models that assume a variable immune removal rate of antibody-bound B. burgdorferi. However, models with a constant immune removal rate could reproduce the rapid population decline of B. burgdorferi populations but not their long-term persistence within hosts using parameter values determined by fitting empirical data. The model predictions, along with the assumptions about the interactions between B. burgdorferi and the immune response, can be tested experimentally to estimate the likelihood that each mechanism affects B. burgdorferi population dynamics in real infections.

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle.

Infections with tick-transmitted Borreliella (Borrelia) burgdorferi, the cause of Lyme disease, represent an increasingly large public health problem in North America and Europe. The ability of these spirochetes to maintain themselves for extended periods of time in their tick vectors and vertebrate reservoirs is crucial for continuance of the enzootic cycle as well as for the increasing exposure of humans to them. The stringent response mediated by the alarmone (p)ppGpp has been determined to be a master regulator in B. burgdorferi. It modulates the expression of identified and unidentified open reading frames needed to deal with and overcome the many nutritional stresses and other challenges faced by the spirochete in ticks and animal reservoirs. The metabolic and morphologic changes resulting from activation of the stringent response in B. burgdorferi may also be involved in the recently described non-genetic phenotypic phenomenon of tolerance to otherwise lethal doses of antimicrobials and to other antimicrobial activities. It may thus constitute a linchpin in multiple aspects of infections with Lyme disease borrelia, providing a link between the micro-ecological challenges of its enzootic life-cycle and long-term residence in the tissues of its animal reservoirs, with the evolutionary side effect of potential persistence in incidental human hosts.