Jon T. Skare

Profiling of temperature-induced changes in Borrelia burgdorferi gene expression by using whole genome arrays.

ABSTRACT Borrelia burgdorferi is the etiologic agent of Lyme disease, the most prevalent arthropod-borne disease in the United States. The genome of the type strain, B31, consists of a 910,725-bp linear chromosome and 21 linear and circular plasmids comprising 610,694 bp. During its life cycle, the spirochete exists in distinctly different environments, cycling between a tick vector and a mammalian host. Temperature is one environmental factor known to affect B. burgdorferi gene expression. To identify temperature-responsive genes, genome arrays containing 1,662 putative B. burgdorferi open reading frames (ORFs) were prepared on nylon membranes and employed to assess gene expression in B. burgdorferi B31 grown at 23 and 35°C. Differences in expression of more than 3.5 orders of magnitude could be readily discerned and quantitated. At least minimal expression from 91% of the arrayed ORFs could be detected. A total of 215 ORFs were differentially expressed at the two temperatures; 133 were expressed at significantly greater levels at 35°C, and 82 were more significantly expressed at 23°C. Of these 215 ORFs, 134 are characterized as genes of unknown function. One hundred thirty-six (63%) of the differentially expressed genes are plasmid encoded. Of particular interest is plasmid lp54 which contains 76 annotated putative genes; 31 of these exhibit temperature-regulated expression. These findings underscore the important role plasmid-encoded genes may play in adjustment of B. burgdorferi to growth under diverse environmental conditions.

The BosR regulatory protein of Borrelia burgdorferi interfaces with the RpoS regulatory pathway and modulates both the oxidative stress response and pathogenic properties of the Lyme disease spirochete.

Borrelia burgdorferi, the Lyme disease spirochete, adapts as it moves between the arthropod and mammalian hosts that it infects. We hypothesize that BosR serves as a global regulator in B. burgdorferi to modulate the oxidative stress response and adapt to mammalian hosts. To test this hypothesis, a bosR mutant in a low‐passage B. burgdorferi isolate was constructed. The resulting bosR::kanR strain was altered when grown microaerobically or anaerobically suggesting that BosR is required for optimal replication under both growth conditions. The absence of BosR increased the sensitivity of B. burgdorferi to hydrogen peroxide and reduced the synthesis of Cdr and NapA, proteins important for cellular redox balance and the oxidative stress response, respectively, suggesting an important role for BosR in borrelial oxidative homeostasis. For the bosR mutant, the production of RpoS was abrogated and resulted in the loss of OspC and DbpA, suggesting that BosR interfaces with the Rrp2–RpoN–RpoS regulatory cascade. Consistent with the linkage to RpoS, cells lacking bosR were non‐infectious in the mouse model of infection. These results indicate that BosR is required for resistance to oxidative stressors and provides a regulatory response that is necessary for B. burgdorferi pathogenesis.

Bioluminescent imaging of Borrelia burgdorferi in vivo demonstrates that the fibronectin-binding protein BBK32 is required for optimal infectivity

The aetiological agent of Lyme disease, Borrelia burgdorferi, is transmitted via infected Ixodes spp. ticks. Infection, if untreated, results in dissemination to multiple tissues and significant morbidity. Recent developments in bioluminescence technology allow in vivo imaging and quantification of pathogenic organisms during infection. Herein, luciferase‐expressing B. burgdorferi and strains lacking the decorin adhesins DbpA and DbpB, as well as the fibronectin adhesin BBK32, were quantified by bioluminescent imaging to further evaluate their pathogenic potential in infected mice. Quantification of bacterial load was verified by quantitative PCR (qPCR) and cultivation. B. burgdorferi lacking DbpA and DbpB were only seen at the 1 h time point post infection, consistent with its low infectivity phenotype. The bbk32 mutant exhibited a significant decrease in its infectious load at day 7 relative to its parent. This effect was most pronounced at lower inocula and imaging correlated well with qPCR data. These data suggest that BBK32‐mediated binding plays an important role in B. burgdorferi colonization. As such, in vivo imaging of bioluminescent Borrelia provides a sensitive means to detect, quantify and temporally characterize borrelial dissemination in a non‐invasive, physiologically relevant environment and, more importantly, demonstrated a quantifiable infectivity defect for the bbk32 mutant.

Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules

Systemic dissemination of microbial pathogens permits microbes to spread from the initial site of infection to secondary target tissues and is responsible for most mortality due to bacterial infections. Dissemination is a critical stage of disease progression by the Lyme spirochaete, Borrelia burgdorferi. However, many mechanistic features of the process are not yet understood. A key step is adhesion of circulating microbes to vascular surfaces in the face of the shear forces present in flowing blood. Using real‐time microscopic imaging of the Lyme spirochaete in living mice we previously identified the first bacterial protein (B. burgdorferi BBK32) shown to mediate vascular adhesion in vivo. Vascular adhesion is also dependent on host fibronectin (Fn) and glycosaminoglycans (GAGs). In the present study, we investigated the mechanisms of BBK32‐dependent vascular adhesion in vivo. We determined that BBK32–Fn interactions (tethering) function as a molecular braking mechanism that permits the formation of more stable BBK32–GAG interactions (dragging) between circulating bacteria and vascular surfaces. Since BBK32‐like proteins are expressed in a variety of pathogens we believe that the vascular adhesion mechanisms we have deciphered here may be critical for understanding the dissemination mechanisms of other bacterial pathogens.

Characterization of a Conditional bosR Mutant in Borrelia burgdorferi

ABSTRACT Borrelia burgdorferi, the etiological agent of Lyme disease, adapts to unique host environments as a consequence of its complex life cycle that spans both arthropod and mammalian species. In this regard, B. burgdorferi must adapt to various environmental signals, pHs, temperatures, and O2 and CO2 levels to establish infectious foci. We hypothesize that the BosR protein functions as a global regulator that is required for both borrelial oxidative homeostasis and pathogenesis. To assess the role of BosR in B. burgdorferi, we constructed an IPTG (isopropyl-β-d-thiogalactopyranoside)-regulated bosR strain. The selective decrease of bosR resulted in a change in growth when cells were cultured either anaerobically or microaerobically; however, a distinct growth defect was observed for anaerobically grown B. burgdorferi relative to the growth attenuation observed for microaerobically grown B. burgdorferi. B. burgdorferi cells in which BosR levels were reduced were more sensitive to hydrogen peroxide and produced lower levels of NapA (Dps) and SodA, proteins involved in the oxidative stress response. In addition, the levels of OspC and DbpA were also induced coincident with increased BosR levels, suggesting that BosR interfaces with the RpoS regulatory cascade, which is known to modulate virulence gene expression in B. burgdorferi. Taken together, these results indicate that BosR is involved in the resistance of B. burgdorferi to oxidative stressors and affects the expression of genes, either directly or indirectly, whose products are important in borrelial pathogenesis.

Invasion of Eukaryotic Cells by Borrelia burgdorferi Requires β1 Integrins and Src Kinase Activity

ABSTRACT Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most widespread tick-borne infection in the northern hemisphere that results in a multistage disorder with concomitant pathology, including arthritis. During late-stage experimental infection in mice, B. burgdorferi evades the adaptive immune response despite the presence of borrelia-specific bactericidal antibodies. In this study we asked whether B. burgdorferi could invade fibroblasts or endothelial cells as a mechanism to model the avoidance from humorally based clearance. A variation of the gentamicin protection assay, coupled with the detection of borrelial transcripts following gentamicin treatment, indicated that a portion of B. burgdorferi cells were protected in the short term from antibiotic killing due to their ability to invade cultured mammalian cells. Long-term coculture of B. burgdorferi with primary human fibroblasts provided additional support for intracellular protection. Furthermore, decreased invasion of B. burgdorferi in murine fibroblasts that do not synthesize the β1 integrin subunit was observed, indicating that β1-containing integrins are required for optimal borrelial invasion. However, β1-dependent invasion did not require either the α5β1 integrin or the borrelial fibronectin-binding protein BBK32. The internalization of B. burgdorferi was inhibited by cytochalasin D and PP2, suggesting that B. burgdorferi invasion required the reorganization of actin filaments and Src family kinases (SFK), respectively. Taken together, these results suggest that B. burgdorferi can invade and retain viability in nonphagocytic cells in a process that may, in part, help to explain the phenotype observed in untreated experimental infection.

Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex

Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems.

Genetic Transformation of Borrelia burgdorferi

The development of robust genetic tools to manipulate Borrelia burgdorferi, the etiologic agent of Lyme disease, now allows investigators to assess the role(s) of individual genes in the context of experimental Lyme borreliosis. This unit is devoted to the description of experimental approaches that are available for the molecular genetic analysis of B. burgdorferi with an emphasis on cultivation, electrotransformation, selection of desired mutants, and genetic complementation of acquired mutants. The intent is to provide a consensus protocol that encapsulates the methodologies currently employed by the B. burgdorferi research community and describe pertinent issues that must be accounted for when working with these pathogenic spirochetal bacteria. Curr. Protoc. Microbiol. 20:12C.4.1‐12C.4.17.

The BBA33 lipoprotein binds collagen and impacts Borrelia burgdorferi pathogenesis

Borrelia burgdorferi, the etiologic agent of Lyme disease, adapts to the mammalian hosts by differentially expressing several genes in the BosR and Rrp2‐RpoN‐RpoS dependent pathways, resulting in a distinct protein profile relative to that seen for survival in the Ixodes spp. tick. Previous studies indicate that a putative lipoprotein, BBA33, is produced in an RpoS‐dependent manner under conditions that mimic the mammalian component of the borrelial lifecycle. However, the significance and function for BBA33 is not known. Given its linkage to the BosR/Rrp2‐RpoN‐RpoS regulatory cascade, we hypothesized that BBA33 facilitates B. burgdorferi infection in the mammalian host. The deletion of bba33 eliminated B. burgdorferi infectivity in C3H mice, which was rescued by genetic complementation with intact bba33. With regard to function, a combinatorial peptide approach, coupled with subsequent in vitro binding assays, indicated that BBA33 binds to collagen type VI and, to a lesser extent, collagen type IV. Whole cell binding assays demonstrated BBA33‐dependent binding to human collagen type VI. Taken together, these results suggest that BBA33 interacts with collagenous structures and may function as an adhesin in a process that is required to prevent bacterial clearance.

Biomechanics of Borrelia burgdorferi Vascular Interactions

SUMMARY Systemic dissemination of microbes is critical for progression of many infectious diseases and is associated with most mortality due to bacterial infection. The physical mechanisms mediating a key dissemination step, bacterial association with vascular endothelia in blood vessels, remain unknown. Here, we show that endothelial interactions of the Lyme disease spirochete Borrelia burgdorferi under physiological shear stress mechanistically resemble selectin-dependent leukocyte rolling. Specifically, these interactions are mediated by transfer of mechanical load along a series of adhesion complexes and are stabilized by tethers and catch bond properties of the bacterial adhesin BBK32. Furthermore, we found that the forces imposed on adhesive bonds under flow may be small enough to permit active migration driven by bacterial flagellar motors. These findings provide insight into the biomechanics of bacterial-vascular interactions and demonstrate that disseminating bacteria and circulating host immune cells share widely conserved mechanisms for interacting with endothelia under physiological shear stress.