What ticks do under your skin: two-photon intravital imaging of Ixodes scapularis feeding in the presence of the lyme disease spirochete.

Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding.

Cultural interpretation on xiang thinking of traditional Chinese medicine.

Though the analysis on the characteristics of Xiang thinking and the cultural background of its formation, it is believed that Xiang thinking is not only an art of logic and thinking with the natural holistic view but also represents the most important cognitive patterns concerning knowledge system of Traditional Chinese Medicine, and even it is the soul of Traditional Chinese Medicine culture. Therefore, a new viewpoint has been proposed that it is necessary to adhere to Xiang thinking with the study of Xiang as a core procedure and to seek for breakthrough for academic innovation in the cognitive process of Xiang with efforts.

The RpoS Gatekeeper in Borrelia burgdorferi: An Invariant Regulatory Scheme That Promotes Spirochete Persistence in Reservoir Hosts and Niche Diversity.

Maintenance of Borrelia burgdorferi within its enzootic cycle requires a complex regulatory pathway involving the alternative σ factors RpoN and RpoS and two ancillary trans-acting factors, BosR and Rrp2. Activation of this pathway occurs within ticks during the nymphal blood meal when RpoS, the effector σ factor, transcribes genes required for tick transmission and mammalian infection. RpoS also exerts a 'gatekeeper' function by repressing σ70-dependent tick phase genes (e.g., ospA, lp6.6). Herein, we undertook a broad examination of RpoS functionality throughout the enzootic cycle, beginning with modeling to confirm that this alternative σ factor is a 'genuine' RpoS homolog. Using a novel dual color reporter system, we established at the single spirochete level that ospA is expressed in nymphal midguts throughout transmission and is not downregulated until spirochetes have been transmitted to a naïve host. Although it is well established that rpoS/RpoS is expressed throughout infection, its requirement for persistent infection has not been demonstrated. Plasmid retention studies using a trans-complemented ΔrpoS mutant demonstrated that (i) RpoS is required for maximal fitness throughout the mammalian phase and (ii) RpoS represses tick phase genes until spirochetes are acquired by a naïve vector. By transposon mutant screening, we established that bba34/oppA5, the only OppA oligopeptide-binding protein controlled by RpoS, is a bona fide persistence gene. Lastly, comparison of the strain 297 and B31 RpoS DMC regulons identified two cohorts of RpoS-regulated genes. The first consists of highly conserved syntenic genes that are similarly regulated by RpoS in both strains and likely required for maintenance of B. burgdorferi sensu stricto strains in the wild. The second includes RpoS-regulated plasmid-encoded variable surface lipoproteins ospC, dbpA and members of the ospE/ospF/elp, mlp, revA, and Pfam54 paralogous gene families, all of which have evolved via inter- and intra-strain recombination. Thus, while the RpoN/RpoS pathway regulates a 'core' group of orthologous genes, diversity within RpoS regulons of different strains could be an important determinant of reservoir host range as well as spirochete virulence.

Two Photon Intravital Microscopy of Lyme Borrelia in Mice.

Two-photon intravital microscopy is a powerful tool that allows visualization of cells in intact tissues in a live animal in real time. In recent years, this advanced technology has been applied to understand pathogen-host interactions using fluorescently labeled bacteria. In particular, infectious fluorescent transformants of the Lyme disease spirochete Borrelia burgdorferi, an Ixodes tick-transmitted pathogen, have been imaged by two-photon intravital microscopy to study bacterial motility and interactions of the pathogen with feeding ticks and host tissues. Here, we describe the techniques and equipment used to image mammalian-adapted spirochetes in the skin of living mice in vivo and in joints ex vivo using two-photon intravital microscopy.

Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone.

Human babesiosis is a tick-borne multisystem disease caused by Babesia species of the apicomplexan phylum. Most clinical cases and fatalities of babesiosis are caused by Babesia microti Current treatment for human babesiosis consists of two drug combinations, atovaquone + azithromycin or quinine + clindamycin. These treatments are associated with adverse side effects and a significant rate of drug failure. Here, we provide evidence for radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone (ELQ) prodrug and atovaquone. In vivo efficacy studies in mice using ELQ-271, ELQ-316, and the ELQ-316 prodrug, ELQ-334, demonstrated excellent growth inhibitory activity against the parasite, with potency equal to that of orally administered atovaquone at 10 mg/kg. Analysis of recrudescent parasites after ELQ or atovaquone monotherapy identified genetic substitutions in the Qi or Qo sites, respectively, of the cytochrome bc1 complex. Impressively, a combination of ELQ-334 and atovaquone, at doses as low as 5.0 mg/kg each, resulted in complete clearance of the parasite with no recrudescence up to 122 d after discontinuation of therapy. These results will set the stage for future clinical evaluation of ELQ and atovaquone combination therapy for treatment of human babesiosis.

Potent and selective inhibitors of Staphylococcus epidermidis tryptophanyl-tRNA synthetase.

OBJECTIVES: The skin commensal and opportunistic pathogen Staphylococcus epidermidis is one of the leading causes of nosocomial and biofilm-associated infections, which urgently requires discovery of new antibiotics. We decided to find new leads that target the S. epidermidis tryptophanyl-tRNA synthetase (SeWRS), which is essential for translation. METHODS: We applied an approach combining structure-based discovery in silico with biochemical and biological experiments in vitro to screen SeWRS inhibitors. RESULTS: Three compounds have an inhibitory effect on enzymatic activities of SeWRS, of which two show low inhibition of the human tryptophanyl-tRNA synthetase. Binding of these compounds to bacterially expressed SeWRS was demonstrated by surface plasmon resonance technology. These three compounds can also obviously inhibit growth of S. epidermidis in vitro and displayed low cytotoxicity to mammalian cells. CONCLUSIONS: These compounds are good leads to develop new antibiotics.

Humoral immunity reflects altered T helper cell bias in Borrelia burgdorferi-infected gamma delta T-cell-deficient mice.

In murine Lyme borreliosis, the absence of gamma delta T lymphocytes augments the T helper cell 2 type humoral response but does not alter disease susceptibility. Arthropod transmission of Borrelia burgdorferi spirochetes results in similar antibody isotypes when gamma delta T cells are present, suggesting that vector effects can negate gamma delta T-cell functions in vivo.

Detection of attenuated, noninfectious spirochetes in Borrelia burgdorferi-infected mice after antibiotic treatment.

Xenodiagnosis by ticks was used to determine whether spirochetes persist in mice after 1 month of antibiotic therapy for vectorborne Borrelia burgdorferi infection. Immunofluorescence and polymerase chain reaction (PCR) were used to show that spirochetes could be found in Ixodes scapularis ticks feeding on 4 of 10 antibiotic-treated mice up to 3 months after therapy. These spirochetes could not be transmitted to naive mice, and some lacked genes on plasmids correlating with infectivity. By 6 months, antibiotic-treated mice no longer tested positive by xenodiagnosis, and cortisone immunosuppression did not alter this result. Nine months after treatment, low levels of spirochete DNA could be detected by real-time PCR in a subset of antibiotic-treated mice. In contrast to sham-treated mice, antibiotic-treated mice did not have culture or histopathologic evidence of persistent infection. These results provide evidence that noninfectious spirochetes can persist for a limited duration after antibiotics but are not associated with disease in mice.