Mari Bonde

Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi

Borrelia burgdorferi has an unusual genomic structure containing 21 plasmids. These plasmids carry genes that are essential for infectivity and survival of the spirochetes in vivo. Several plasmids are lost during cultivation in vitro, which might lead to a heterogeneous population after multiple passages and loss of infectivity in laboratory animals. Herein, we present a simple and inexpensive multiplex PCR method that detects the complete plasmid profile of B. burgdorferi B31 in just two PCR tubes.

P66 porins are present in both Lyme disease and relapsing fever spirochetes : a comparison of the biophysical properties of P66 porins from six Borrelia species

The genus Borrelia is the cause of the two human diseases: Lyme disease (LD) and relapsing fever (RF). Both LD and RF Borrelia species are obligate parasites and are dependent on nutrients provided by their hosts. The first step of nutrient uptake across the outer membrane of these Gram-negative bacteria is accomplished by water-filled channels, so-called porins. The knowledge of the porin composition in the outer membranes of the different pathogenic Borrelia species is limited. Only one porin has been described in relapsing fever spirochetes to date, whereas four porins are known to be present in Lyme disease agents. From these, the Borrelia burgdorferi outer membrane channel P66 is known to act as an adhesin and was well studied as a porin. To investigate if P66 porins are expressed and similarly capable of pore formation in other Borrelia causing Lyme disease or relapsing fever three LD species (B. burgdorferi, B. afzelii, B. garinii) and three RF species (B. duttonii, B. recurrentis and B. hermsii) were investigated for outer membrane proteins homologous to P66. A search in current published RF genomes, comprising the ones of B. duttonii, B. recurrentis and B. hermsii, indicated that they all contained P66 homologues. The P66 homologues of the six Borrelia species were purified to homogeneity and their pore-forming abilities as well as the biophysical properties of the pores were analyzed using the black lipid bilayer assay.

Integrin binding by Borrelia burgdorferi P66 facilitates dissemination but is not required for infectivity.

P66, a Borrelia burgdorferi surface protein with porin and integrin‐binding activities, is essential for murine infection. The role of P66 integrin‐binding activity in B. burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202–208). Neither change affected surface localization or channel‐forming activity of P66, but both significantly reduced binding to αvβ3. Integrin‐binding deficient B. burgdorferi strains caused disseminated infection in mice at 4 weeks post‐subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202–208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post‐inoculation, but bacterial burdens recovered to wild‐type levels at 4 weeks post‐inoculation. The delay in tissue colonization correlated with reduced migration of the Del202–208 strains across microvascular endothelial cells, similar to Δp66 bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.

Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than ‘normal’ Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80–90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.

DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates

Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin.

Background: B. burgdorferi P13 has unique characteristics compared with other porins. Results: P13 is a homo-oligomer with a 0.6 nS conductance in 1 m KCl and a substrate cut-off of 400 Da. Conclusion: P13 represents a general diffusion pathway for small solutes into Borrelia. Significance: Understanding the molecular transport through P13 may play a role in designing more efficient antibiotic treatments against Borrelia infections. P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

Metabolic Signature Profiling as a Diagnostic and Prognostic Tool in Pediatric Plasmodium falciparum Malaria

The metabolic profile in paediatric patients suffering from acute P. falciparum malaria carries sufficient information to grade disease severity.

The oxylipin and endocannabidome responses in acute phase Plasmodium falciparum malaria in children.

BackgroundOxylipins and endocannabinoids are low molecular weight bioactive lipids that are crucial for initiation and resolution of inflammation during microbial infections. Metabolic complications in malaria are recognized contributors to severe and fatal malaria, but the impact of malaria infection on the production of small lipid derived signalling molecules is unknown. Knowledge of immunoregulatory patterns of these molecules in malaria is of great value for better understanding of the disease and improvement of treatment regimes, since the action of these classes of molecules is directly connected to the inflammatory response of the organism.MethodsDetection of oxylipins and endocannabinoids from plasma samples from forty children with uncomplicated and severe malaria as well as twenty controls was done after solid phase extraction followed by chromatography mass spectrometry analysis. The stable isotope dilution method was used for compound quantification. Data analysis was done with multivariate (principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA®) and univariate approaches (receiver operating characteristic (ROC) curves, t tests, correlation analysis).ResultsForty different oxylipin and thirteen endocannabinoid metabolites were detected in the studied samples, with one oxylipin (thromboxane B2, TXB2) in significantly lower levels and four endocannabinoids (OEA, PEA, DEA and EPEA) at significantly higher levels in infected individuals as compared to controls according to t test analysis with Bonferroni correction. Three oxylipins (13-HODE, 9-HODE and 13-oxo-ODE) were higher in severe compared to uncomplicated malaria cases according to the results from multivariate analysis. Observed changes in oxylipin levels can be connected to activation of cytochrome P450 (CYP) and 5-lipoxygenase (5-LOX) metabolic pathways in malaria infected individuals compared to controls, and related to increased levels of all linoleic acid oxylipins in severe patients compared to uncomplicated ones. The endocannabinoids were extremely responsive to malaria infection with majority of this class of molecules found at higher levels in infected individuals compared to controls.ConclusionsIt was possible to detect oxylipin and endocannabinoid molecules that can be potential biomarkers for differentiation between malaria infected individuals and controls and between different classes of malaria. Metabolic pathways that could be targeted towards an adjunctive therapy in the treatment of malaria were also pinpointed.