Lyme Disease Abstracts 1

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A manganese-rich environment supports superoxide dismutase activity in a Lyme disease pathogen, Borrelia burgdorferi.
            (Aguirre et al., 2013) Download
The Lyme disease pathogen Borrelia burgdorferi represents a novel organism in which to study metalloprotein biology in that this spirochete has uniquely evolved with no requirement for iron. Not only is iron low, but we show here that B. burgdorferi has the capacity to accumulate remarkably high levels of manganese. This high manganese is necessary to activate the SodA superoxide dismutase (SOD) essential for virulence. Using a metalloproteomic approach, we demonstrate that a bulk of B. burgdorferi SodA directly associates with manganese, and a smaller pool of inactive enzyme accumulates as apoprotein. Other metalloproteins may have similarly adapted to using manganese as co-factor, including the BB0366 aminopeptidase. Whereas B. burgdorferi SodA has evolved in a manganese-rich, iron-poor environment, the opposite is true for Mn-SODs of organisms such as Escherichia coli and bakers' yeast. These Mn-SODs still capture manganese in an iron-rich cell, and we tested whether the same is true for Borrelia SodA. When expressed in the iron-rich mitochondria of Saccharomyces cerevisiae, B. burgdorferi SodA was inactive. Activity was only possible when cells accumulated extremely high levels of manganese that exceeded cellular iron. Moreover, there was no evidence for iron inactivation of the SOD. B. burgdorferi SodA shows strong overall homology with other members of the Mn-SOD family, but computer-assisted modeling revealed some unusual features of the hydrogen bonding network near the enzyme's active site. The unique properties of B. burgdorferi SodA may represent adaptation to expression in the manganese-rich and iron-poor environment of the spirochete.

Vitamin A deficiency exacerbates murine Lyme arthritis
            (Cantorna and Hayes, 1996) Download
Vitamin A deficiency predisposes the host for a strong inflammatory response, suggesting that it may foster susceptibility to diseases, such as Lyme arthritis, in which activated macrophage and inflammatory cytokine production are pathogenic. Infected mice had a rapid serum retinal decline that correlated with the onset of arthritis. The mice with the least retinol developed acute arthritis earlier and more severely than those with the highest retinol. Earlier and stronger interleukin (IL)-12, interferon-gamma (IFN)-gamma, and tumor necrosis factor responses were found in Borrelia burgdorferi-infected, vitamin A-deficient mice compared with controls. The spirochetes induced IFN-gamma secretion from unprimed cells, and retinoid addition in vitro inhibited IFN-gamma synthesis. Vitamin A deficiency may exacerbate acute Lyme arthritis by enhancing an acute arthritogenic inflammatory response initiated by spirochete-driven IFN-gamma secretion. Conversely, vitamin A may lessen acute Lyme arthritis pathology by blocking IFN-gamma and IL-12 synthesis.

Selective Essential Oils from Spice or Culinary Herbs Have High Activity against Stationary Phase and Biofilm Borrelia burgdorferi.
            (Feng et al., 2017) Download
Although the majority of patients with acute Lyme disease can be cured with the standard 2-4 week antibiotic treatment, about 10-20% of patients continue suffering from chronic symptoms described as posttreatment Lyme disease syndrome. While the cause for this is debated, one possibility is that persister bacteria are not killed by the current Lyme antibiotics and remain active in the system. It has been reported that essential oils have antimicrobial activities and some have been used by patients with persisting Lyme disease symptoms. However, the activity of essential oils against the causative agent Borrelia burgdorferi (B. burgdorferi) has not been well studied. Here, we evaluated the activity of 34 essential oils against B. burgdorferi stationary phase culture as a model for persister bacteria. We found that not all essential oils had activity against the B. burgdorferi stationary phase culture, with top five essential oils (oregano, cinnamon bark, clove bud, citronella, and wintergreen) at a low concentration of 0.25% showing high anti-persister activity that is more active than the known persister drug daptomycin. Interestingly, some highly active essential oils were found to have excellent anti-biofilm ability as shown by their ability to dissolve the aggregated biofilm-like structures. The top three hits, oregano, cinnamon bark, and clove bud completely eradicated all viable cells without any regrowth in subculture in fresh medium, whereas but not citronella and wintergreen did not have this effect. Carvacrol was found to be the most active ingredient of oregano oil showing excellent activity against B. burgdorferi stationary phase cells, while other ingredients of oregano oil p-cymene and α-terpinene had no apparent activity. Future studies are needed to characterize and optimize the active essential oils in drug combination studies in vitro and in vivo and to address their safety and pharmacokinetic properties before they can be considered as a novel treatment of persistent Lyme disease.


 

Identification of Essential Oils with Strong Activity against Stationary Phase Borrelia burgdorferi.
            (Feng et al., 2018) Download
Lyme disease is the most common vector borne-disease in the United States (US). While the majority of the Lyme disease patients can be cured with 2⁻4 weeks antibiotic treatment, about 10⁻20% of patients continue to suffer from persisting symptoms. While the cause of this condition is unclear, persistent infection was proposed as one possibility. It has recently been shown that B. burgdorferi develops dormant persisters in stationary phase cultures that are not killed by the current Lyme antibiotics, and there is interest in identifying novel drug candidates that more effectively kill such forms. We previously identified some highly active essential oils with excellent activity against biofilm and stationary phase B. burgdorferi. Here, we screened another 35 essential oils and found 10 essential oils (Allium sativum L. bulbs, Pimenta officinalis Lindl. berries, Cuminum cyminum L. seeds, Cymbopogon martini var. motia Bruno grass, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers, Amyris balsamifera L. wood, Thymus vulgaris L. leaves, Litsea cubeba (Lour.) Pers. fruits, Eucalyptus citriodora Hook. leaves) and the active component of cinnamon bark cinnamaldehyde (CA) at a low concentration of 0.1% have strong activity against stationary phase B. burgdorferi. At a lower concentration of 0.05%, essential oils of Allium sativum L. bulbs, Pimenta officinalis Lindl. berries, Cymbopogon martini var. motia Bruno grass and CA still exhibited strong activity against the stationary phase B. burgdorferi. CA also showed strong activity against replicating B. burgdorferi, with a MIC of 0.02% (or 0.2 μg/mL). In subculture studies, the top five essential oil hits Allium sativum L. bulbs, Pimenta officinalis Lindl. berries, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers, and Litsea cubeba (Lour.) Pers. fruits completely eradicated all B. burgdorferi stationary phase cells at 0.1%, while Cymbopogon martini var. motia Bruno grass, Eucalyptus citriodora Hook. leaves, Amyris balsamifera L. wood, Cuminum cyminum L. seeds, and Thymus vulgaris L. leaves failed to do so as shown by visible spirochetal growth after 21-day subculture. At concentration of 0.05%, only Allium sativum L. bulbs essential oil and CA sterilized the B. burgdorferi stationary phase culture, as shown by no regrowth during subculture, while Pimenta officinalis Lindl. berries, Commiphora myrrha (T. Nees) Engl. resin, Hedychium spicatum Buch.-Ham. ex Sm. flowers and Litsea cubeba (Lour.) Pers. fruits essential oils all had visible growth during subculture. Future studies are needed to determine if these highly active essential oils could eradicate persistent B. burgdorferi infection in vivo.


 

A prospective cross-over field trial shows protection of lemon eucalyptus extract against tick bites
            (Gardulf et al., 2004) Download
Ixodes ricinus can transmit several microorganisms, out of which Borrelia burgdorferi and tick-borne encephalitis (TBE) virus are the most important pathogens in humans. A lemon eucalyptus extract (Citriodiol) has been shown to be a natural repellent against mosquitoes, stable flies, and midges. We have investigated whether the use of the extract would reduce the number of attached I. ricinus ticks in humans. A volunteer sample of 111 individuals was recruited among healthy, outdoor active adults living in two highly I. ricinus-infested areas. They were randomly divided into two groups: one group applied the Citriodiol spray daily for 2 wk to the lower extremities, whereas the other group was instructed not to use any type of repellent on any part of the body. After 2 wk, the scheme was reversed. All volunteers filled in a daily diary, giving information about outdoor activities and the number of observed, attached, or not yet attached ticks, including the anatomical location of the ticks found. Forty-two attached ticks were reported during the weeks when the Citriodiol spray was used, and 112 were reported when it was not. The median number of reported attached ticks per person decreased from 1.5 (range, 0-9) to 0.5 (range, 0-2; P < 0.05) during the weeks when the repellent was used. The number of reported attached ticks noted below the waist was 13/42 (31%) during the period when the spray was used and 73/112 (65%) when no spray was used (P < 0.001). No evident redistribution of attached ticks from protected areas to unprotected areas was seen. Citriodiol may become a useful means in reducing the number of tick bites and thereby tick-borne infections, although additional studies are warranted.

Anti-borreliae efficacy of selected organic oils and fatty acids.
            (Goc et al., 2019) Download
BACKGROUND:  Borrelia sp. is a causative pathogen of Lyme disease which has become a worldwide health concern. Non-toxic approaches especially directed toward latent persistent forms of this pathogen are desired. Lipids in the form of volatile and non-volatile oils, and fatty acids with proven anti-borreliae efficacy could become an additional support or an alternative for consideration in treatment approaches. METHODS:  In this study we investigated 47 lipids (30 volatile and non-volatile oils, and 17 fatty acids) of plant and animal origin against typical motile, knob/round-shaped persisters, and biofilm-like aggregates of Borrelia burgdorferi s.s. and Borrelia garinii, which are identified as pathogenic factors of Lyme disease in the USA and Europe, using direct microscopic counting and spectrofluorometric measurements. RESULTS:  Out of all examined lipids, 5 oils (Bay leaf oil, Birch oil, Cassia oil, Chamomile oil German, and Thyme oil) at or below 0.25%, and 3 fatty acids (13Z,16Z Docosadienoic acid, erucic acid, and petroselinic acid) at or below 0.75 mg/ml, showed bactericidal activity against typical motile spirochetes and knob/round-shaped persisters. Only Bay leaf oil and Cassia oil, including their major constituents, eugenol and cinnamaldehyde, showed to target biofilm-like aggregates of both tested Borrelia spp. at the same concentration, although with 20-30% eradication mark. CONCLUSION:  Based on obtained results, volatile oils were more potent than non-volatile oils, and unsaturated fatty acids were more effective than saturated fatty acids. Among all tested oils, Bay leaf oil and Cassia oil, with their major components eugenol and cinnamaldehyde, seem to have the highest anti-borreliae efficacy.

Role of glutathione metabolism in host defense against Borrelia burgdorferi infection.
            (Kerstholt et al., 2018) Download
Pathogen-induced changes in host cell metabolism are known to be important for the immune response. In this study, we investigated how infection with the Lyme disease-causing bacterium Borrelia burgdorferi (Bb) affects host metabolic pathways and how these metabolic pathways may impact host defense. First, metabolome analysis was performed on human primary monocytes from healthy volunteers, stimulated for 24 h with Bb at low multiplicity of infection (MOI). Pathway analysis indicated that glutathione (GSH) metabolism was the pathway most significantly affected by Bb Specifically, intracellular levels of GSH increased on average 10-fold in response to Bb exposure. Furthermore, these changes were found to be specific, as they were not seen during stimulation with other pathogens. Next, metabolome analysis was performed on serum samples from patients with early-onset Lyme disease in comparison with patients with other infections. Supporting the in vitro analysis, we identified a cluster of GSH-related metabolites, the γ-glutamyl amino acids, specifically altered in patients with Lyme disease, and not in other infections. Lastly, we performed in vitro experiments to validate the role for GSH metabolism in host response against Bb. We found that the GSH pathway is essential for Bb-induced cytokine production and identified glutathionylation as a potential mediating mechanism. Taken together, these data indicate a central role for the GSH pathway in the host response to Bb GSH metabolism and glutathionylation may therefore be important factors in the pathogenesis of Lyme disease and potentially other inflammatory diseases as well.

Short-Chain Fatty Acids Alter Metabolic and Virulence Attributes of Borrelia burgdorferi.
            (Lin et al., 2018) Download
Borrelia burgdorferi responds to a variety of host-derived factors and appropriately alters its gene expression for adaptation under different host-specific conditions. We previously showed that various levels of acetate, a short-chain fatty acid (SCFA), altered the protein profile of B. burgdorferi In this study, we determined the effects of other physiologically relevant SCFAs in the regulation of metabolic/virulence-associated proteins using mutant borrelial strains. No apparent increase in the synthesis of outer surface protein C (OspC) was noted when a carbon storage regulator A (csrA of B. burgdorferi, or csrABb ) mutant (mt) was propagated within dialysis membrane chambers implanted within rat peritoneal cavity, while the parental wild type (wt; B31-A3 strain) and csrABb cis-complemented strain (ct) had increased OspC with a reciprocal reduction in OspA levels. Growth rates of wt, mt, ct, 7D (csrABb mutant lacking 7 amino acids at the C terminus), and 8S (csrABb with site-specific changes altering its RNA-binding properties) borrelial strains were similar in the presence of acetate. Increased levels of propionate and butyrate reduced the growth rates of all strains tested, with mt and 8S exhibiting profound growth deficits at higher concentrations of propionate. Transcriptional levels of rpoS and ospC were elevated on supplementation of SCFAs compared to those of untreated spirochetes. Immunoblot analysis revealed elevated levels of RpoS, OspC, and DbpA with increased levels of SCFAs. Physiological levels of SCFAs prevalent in select human and rodent fluids were synergistic with mammalian host temperature and pH to increase the levels of aforementioned proteins, which could impact the colonization of B. burgdorferi during the mammalian phase of infection.

Manganese and zinc regulate virulence determinants in Borrelia burgdorferi.
            (Troxell et al., 2013) Download
Borrelia burgdorferi, the causative agent of Lyme disease, must adapt to two diverse niches, an arthropod vector and a mammalian host. RpoS, an alternative sigma factor, plays a central role in spirochetal adaptation to the mammalian host by governing expression of many genes important for mammalian infection. B. burgdorferi is known to be unique in metal utilization, and little is known of the role of biologically available metals in B. burgdorferi. Here, we identified two transition metal ions, manganese (Mn(2+)) and zinc (Zn(2+)), that influenced regulation of RpoS. The intracellular Mn(2+) level fluctuated approximately 20-fold under different conditions and inversely correlated with levels of RpoS and the major virulence factor OspC. Furthermore, an increase in intracellular Mn(2+) repressed temperature-dependent induction of RpoS and OspC; this repression was overcome by an excess of Zn(2+). Conversely, a decrease of intracellular Mn(2+) by deletion of the Mn(2+) transporter gene, bmtA, resulted in elevated levels of RpoS and OspC. Mn(2+) affected RpoS through BosR, a Fur family homolog that is required for rpoS expression: elevated intracellular Mn(2+) levels greatly reduced the level of BosR protein but not the level of bosR mRNA. Thus, Mn(2+) and Zn(2+) appeared to be important in modulation of the RpoS pathway that is essential to the life cycle of the Lyme disease spirochete. This finding supports the emerging notion that transition metals such as Mn(2+) and Zn(2+) play a critical role in regulation of virulence in bacteria.


 

Herb⁻Drug Interaction Potential of Anti-Borreliae Effective Extracts from Uncaria tomentosa (Samento) and Otoba parvifolia (Banderol) Assessed In Vitro.
            (Weiss, 2018) Download
Samento (extract from Uncaria tomentosa) and Banderol (extract from Otoba parvifolia) have been demonstrated to have anti-inflammatory and antimicrobial properties, e.g., against different morphological forms of Borrelia burgdorferi. However, there is hardly any data on the pharmacological safety of these two herbal medicines. This in vitro study aimed at scrutinizing their possible characteristics as perpetrators in pharmacokinetic herbal⁻drug interactions. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits and inhibition of drug transporters by use of fluorescent probe substrates. Induction was quantified by real-time RT-PCR and activation of pregnane x receptor (PXR) and aryl hydrocarbon receptor (AhR) by reporter gene assays. Organic anion transporting polypeptide 1B1 (OATP1B1) (IC50 = 0.49 ± 0.28%) and OATP1B3 (IC50 = 0.65 ± 0.29%) were potently inhibited by Banderol, but only weakly by Samento. CYP3A4 was inhibited about 40% at a Samento concentration of 1%. Samento significantly induced mRNA expression of CYP2J2, UGT1A3, UGT1A9, ABCB1, and SLCO1B1 and strongly activated PXR, but hardly AhR. In conclusion, the perpetrator profiles of Samento and Banderol for herb⁻drug interactions completely differ. Clinical studies are strongly recommended to clarify whether the effects observed in vitro are of clinical relevance.

 


References

Aguirre, JD, et al. (2013), ‘A manganese-rich environment supports superoxide dismutase activity in a Lyme disease pathogen, Borrelia burgdorferi.’, J Biol Chem, 288 (12), 8468-78. PubMed: 23376276
Cantorna, M. T. and C. E. Hayes (1996), ‘Vitamin A deficiency exacerbates murine Lyme arthritis’, J Infect Dis, 174 (4), 747-51. PubMed: 8843212
Feng, J, et al. (2017), ‘Selective Essential Oils from Spice or Culinary Herbs Have High Activity against Stationary Phase and Biofilm Borrelia burgdorferi.’, Front Med (Lausanne), 4 169. PubMed: 29075628
Feng, J, et al. (2018), ‘Identification of Essential Oils with Strong Activity against Stationary Phase Borrelia burgdorferi.’, Antibiotics (Basel), 7 (4), PubMed: 30332754
Gardulf, A., I. Wohlfart, and R. Gustafson (2004), ‘A prospective cross-over field trial shows protection of lemon eucalyptus extract against tick bites’, J Med Entomol, 41 (6), 1064-67. PubMed: 15605645
Goc, A, A Niedzwiecki, and M Rath (2019), ‘Anti-borreliae efficacy of selected organic oils and fatty acids.’, BMC Complement Altern Med, 19 (1), 40. PubMed: 30717726
Kerstholt, M, et al. (2018), ‘Role of glutathione metabolism in host defense against Borrelia burgdorferi infection.’, Proc Natl Acad Sci U S A, 115 (10), E2320-28. PubMed: 29444855
Lin, YH, et al. (2018), ‘Short-Chain Fatty Acids Alter Metabolic and Virulence Attributes of Borrelia burgdorferi.’, Infect Immun, 86 (9), PubMed: 29891543
Troxell, B, et al. (2013), ‘Manganese and zinc regulate virulence determinants in Borrelia burgdorferi.’, Infect Immun, 81 (8), 2743-52. PubMed: 23690398
Weiss, J (2018), ‘Herb⁻Drug Interaction Potential of Anti-Borreliae Effective Extracts from Uncaria tomentosa (Samento) and Otoba parvifolia (Banderol) Assessed In Vitro.’, Molecules, 24 (1), PubMed: 30602711