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      Comparative molecular analyses of Borrelia burgdorferi sensu stricto strains B31 and N40D10/E9 and determination of their pathogenicity

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          Abstract

          Background

          Lyme disease in the United States is caused primarily by B. burgdorferi sensu stricto while other species are also prevalent in Europe. Genetic techniques have identified several chromosomal and plasmid-borne regulatory and virulence factors involved in Lyme pathogenesis. B31 and N40 are two widely studied strains of B. burgdorferi, which belong to two different 16 S-23 S rRNA spacer types (RST) and outer surface protein C (OspC) allelic groups. However, the presence of several known virulence factors in N40 has not been investigated. This is the first comprehensive study that compared these two strains both in vitro and using the mouse model of infection.

          Results

          Phylogenetic analyses predict B31 to be more infectious. However, our studies here indicate that N40D10/E9 is more infectious than the B31 strain at lower doses of inoculation in the susceptible C3H mice. Based-upon a careful analyses of known adhesins of these strains, it is predicted that the absence of a known fibronectin-glycosaminoglycan binding adhesin, bbk32, in the N40 strain could at least partially be responsible for reduction in its binding to Vero cells in vitro. Nevertheless, this difference does not affect the infectivity of N40D10/E9 strain. The genes encoding known regulatory and virulence factors critical for pathogenesis were detected in both strains. Differences in the protein profiles of these B. burgdorferi strains in vitro suggest that the novel, differentially expressed molecules may affect infectivity of B. burgdorferi. Further exacerbation of these molecular differences in vivo could affect the pathogenesis of spirochete strains.

          Conclusion

          Based upon the studies here, it can be predicted that N40D10/E9 disseminated infection at lower doses may be enhanced by its lower binding to epithelial cells at the site of inoculation due to the absence of BBK32. We suggest that complete molecular analyses of virulence factors followed by their evaluation using the mouse infection model should form the basis of determining infectivity and pathogenicity of different strains rather than simple phylogenetic group analyses. This study further emphasizes a need to investigate multiple invasive strains of B. burgdorferi to fully appreciate the pathogenic mechanisms that contribute to Lyme disease manifestations.

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          Most cited references 113

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          High resolution two-dimensional electrophoresis of proteins.

           P H O'Farrell (1975)
          A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-diminsional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10 minus 4 to 10 minus 5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.
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            Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi.

            The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.
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              Permanent cell line expressing human factor VIII-related antigen established by hybridization.

              A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line.
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                Author and article information

                Contributors
                Journal
                BMC Microbiol
                BMC Microbiol
                BMC Microbiology
                BioMed Central
                1471-2180
                2012
                30 July 2012
                : 12
                : 157
                Affiliations
                [1 ]Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 225 Warren Street, Newark, NJ 07103–3535, USA
                [2 ]Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California Davis, One Shields Avenue, Davis, CA, 95616
                Article
                1471-2180-12-157
                10.1186/1471-2180-12-157
                3511255
                22846633
                Copyright ©2012 Chan et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Research Article

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