Lyme disease in the United States is most often caused by Borrelia burgdorferi sensu stricto. After a tick bite, the patient may develop erythema migrans at that site.

Removal of particulate materials that would otherwise cumulate within the airspace and hinder the gas exchange is one of the central processes of maintaining lung homeostasis. While the importance of the particle uptake by alveolar macrophages and their expulsion via the airways mucociliary escalator is well established, very little is known about the alternative route for removing the particles via direct crossing the lung epithelium for transfer into the pulmonary lymph and bloodstream. This study dissected sequential mechanisms involved in nanoparticle transcytosis through the alveolar epithelial cell layer. By a combination of live cell, super resolution, and electron microscopy and RNA interference study, we have dissected temporal steps of nanoparticle transcytosis through alveolar epithelium. Our study revealed that caveolin is essential for the firm adhesion of the silica nanoparticle agglomerates to the apical membrane and their subsequent rapid internalization with the help of macropinocytic elements C-terminal-binding protein1 and Rabankyrin-5 but not dynamin. Actin, but not microtubules, played a major role in nanoparticle uptake and subsequent transportation. The compartments with nanoparticles were tethered to trans-Golgi network to be jointly transported along actin stress fibers across the cytoplasm, employing a myosin-dependent mechanism. The trans-Golgi nanoparticle transport machinery was positive to Rab6A, a marker linked to vesicle exocytosis. Exocytosis was primarily occurring at the basolateral plane of the alveolar epithelial cells. The high-proficiency novel caveolin and Rabankyrin-5 associated uptake and transcellular transport of nanoparticles across the AEC barrier supports its importance in clearance of amorphous silica and other types of non-inflammatory nanoparticles that are rapidly removed from the lungs following their inhalation.

Natural killer (NK) cells use the activating receptor NKp30 as a microbial pattern-recognition receptor to recognize, activate cytolytic pathways, and directly kill the fungi Cryptococcus neoformans and Candida albicans. However, the fungal pathogen-associated molecular pattern (PAMP) that triggers NKp30-mediated killing remains to be identified. Here we show that β-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that β-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, β-1,3-glucan increases expression and clustering of NKp30 at the microbial and NK cell synapse to induce perforin release for fungal cytotoxicity. Rather than blocking the interaction between fungi and NK cells, soluble β-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating β-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity.

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between reservoirs and hosts by ticks of the family Ixodidae. Infection with B. burgdorferi in humans causes Lyme disease or Lyme borreliosis. Currently, 20 Lyme disease‐associated Borrelia species and more than 20 relapsing fever‐associated Borrelia species have been described. Identification and differentiation of different Borrelia species and strains is largely dependent on analyses of their genetic characteristics. A variety of molecular techniques have been described for Borrelia isolate speciation, molecular epidemiology, and pathogenicity studies. In this unit, we focus on three basic protocols, PCR‐RFLP‐based typing of the rrs‐rrlA and rrfA‐rrlB ribosomal spacer, ospC typing, and MLST. These protocols can be employed alone or in combination for characterization of B. burgdorferi isolates or directly on uncultivated organisms in ticks, mammalian host reservoirs, and human clinical specimens. Curr. Protoc. Microbiol . 34:12C.1‐12C.31. © 2014 by John Wiley & Sons, Inc.

BACKGROUND:Erythema migrans is the most common manifestation of Lyme disease. Recurrences are not uncommon, and although they are usually attributed to reinfection rather than relapse of the original infection, this remains somewhat controversial. We used molecular typing of Borrelia burgdorferi isolates obtained from patients with culture-confirmed episodes of erythema migrans to distinguish between relapse and reinfection.METHODS:We determined the genotype of the gene encoding outer-surface protein C (ospC) of B. burgdorferi strains detected in cultures of skin or blood specimens obtained from patients with consecutive episodes of erythema migrans. After polymerase-chain-reaction amplification, ospC genotyping was performed by means of reverse line-blot analysis or DNA sequencing of the nearly full-length gene. Most strains were further analyzed by determining the genotype according to the 16S-23S ribosomal RNA intergenic spacer type, multilocus sequence typing, or both. Patients received standard courses of antibiotics for erythema migrans.RESULTS:B. burgdorferi isolates obtained from 17 patients who received a diagnosis of erythema migrans between 1991 and 2011 and who had 22 paired episodes of this lesion (initial and second episodes) were available for testing. The ospC genotype was found to be different at each initial and second episode. Apparently identical genotypes were identified on more than one occasion in only one patient, at the first and third episodes, 5 years apart, but different genotypes were identified at the second and fourth episodes.CONCLUSIONS:None of the 22 paired consecutive episodes of erythema migrans were associated with the same strain of B. burgdorferi on culture. Our data show that repeat episodes of erythema migrans in appropriately treated patients were due to reinfection and not relapse. (Funded by the National Institutes of Health and the William and Sylvia Silberstein Foundation.).

The clinical manifestations of Lyme disease, caused by Borrelia burgdorferi, vary considerably in different patients, possibly due to infection by strains with varying pathogenicity. Both rRNA intergenic spacer and ospC typing methods have proven to be useful tools for categorizing B. burgdorferi strains that vary in their tendency to disseminate in humans. Neither method, however, is suitable for inferring intraspecific relationships among strains that are important for understanding the evolution of pathogenicity and the geographic spread of disease. In this study, multilocus sequence typing (MLST) was employed to investigate the population structure of B. burgdorferi recovered from human Lyme disease patients. A total of 146 clinical isolates from patients in New York and Wisconsin were divided into 53 sequence types (STs). A goeBURST analysis, that also included previously published STs from the northeastern and upper Midwestern US and adjoining areas of Canada, identified 11 major and 3 minor clonal complexes, as well as 14 singletons. The data revealed that patients from New York and Wisconsin were infected with two distinct, but genetically and phylogenetically closely related, populations of B. burgdorferi. Importantly, the data suggest the existence of B. burgdorferi lineages with differential capabilities for dissemination in humans. Interestingly, the data also indicate that MLST is better able to predict the outcome of localized or disseminated infection than is ospC typing.

The persistence of dormant, noncultivable Borrelia burgdorferi after ceftriaxone treatment was examined. B. burgdorferi isolates were cultivated in Barbour-Stoenner-Kelly medium in the presence or absence of ceftriaxone, and cultures were monitored for up to 56 days. Viability of B. burgdorferi was assessed by subculture, growth, morphology, and pH (as a surrogate for metabolic activity). In addition, the presence of B. burgdorferi DNA and mRNA was assayed by PCR and by real-time reverse transcription (RT)-PCR, respectively. Spirochetes could not be successfully subcultured by day 3 after exposure to ceftriaxone. In cultures treated with ceftriaxone, the pH of the culture medium did not change through day 56, whereas it declined by at least 1 pH unit by 14 days in untreated cultures. These results suggest that B. burgdorferi viability is rapidly eliminated after antibiotic treatment. Nevertheless, DNA was detected by B. burgdorferi-specific PCR for up to 56 days in aliquots from both ceftriaxone-treated and untreated cultures. In addition, although ceftriaxone treatment resulted in a reduction in the quantities of transcript for ospC, ospA, flaB, and pfk, certain mRNAs could be detected through day 14. Transcript for all 4 genes was essentially undetectable after 28 days of treatment. Taken together, the results suggest that B. burgdorferi DNA and mRNA can be detected in samples long after spirochetes are no longer viable as assessed by classic microbiological parameters. PCR positivity in the absence of culture positivity following antibiotic treatment in animal and human studies should be interpreted with caution.