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

Chan et al. BMC Microbiology 0 Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California Davis , One Shields Avenue, Davis, CA95616 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 Chan et al. - Comparative molecular analyses of Borrelia burgdorferi sensu stricto strains B31 and N40D10/ E9 and determination of their pathogenicity R E S E A R C H A R T I C L E Comparative molecular analyses of Borrelia burgdorferi sensu stricto strains B31 and N40D10/ E9 and determination of their pathogenicity Kamfai Chan1, Mehwish Awan1, Stephen W Barthold2 and Nikhat Parveen1* 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 fibronectinglycosaminoglycan 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. Open Access * Correspondence: 1Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 225 Warren Street, Newark, NJ 071033535, USA Full list of author information is available at the end of the article Background Lyme disease is a multisystemic disease caused by Borrelia burgdorferi, which is transmitted by Ixodes ticks in the United States of America [1,2]. The earliest clinical sign of Lyme disease is an expanding rash at the site of tick bite known as erythema migrans [3]. If left untreated, infection with Lyme spirochetes can disseminate to joints, heart, skin and central nervous system [3]. A resulting persistent infection of the host can then result in the development of arthritis, carditis, or neuroborreliosis [4]. Arthritis is the primary manifestation of late and chronic Lyme disease by B. burgdorferi sensu stricto, the predominant genospecies in the United States. The genetic basis of bacterial virulence and disease has been investigated in a large number of Gramnegative and Gram-positive bacteria in the last three decades and major virulence factors of each microbe have been identified. These studies have shown that various strains of bacterial pathogens often exhibit different levels of pathogenicity and disease manifestations in the hosts. In most cases, the high pathogenicity is associated with specific variations in the set of virulence factors [5-11]. In many microbes, the respective virulence factor-encoding genes are clustered together in specific regions defined as pathogenicity islands [12]. Strains of B. burgdorferi show a high variation in their ability to cause disseminated infection. Since genetic studies have been developed in this spirochete only in the past decade, classification based upon its virulence factor diversity has not yet been fully developed. Furthermore, the presence of a segmented genome has hampered studies with different spirochete strains. However, B. burgdorferi sensu stricto strains have been divided into different groups either on the basis of allelic variation in the Outer surface protein C (OspC), which is essential for causing infection in the mammalian hosts [13-16], or the polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of 16 S-23 S rRNA spacer types (RST). Furthermore, ospC or RST groups were used as markers to determine pathogenicity of different B. burgdorferi strains with only some groups considered invasive [17-24]. Studies involving the two most widely investigated strains, B31 and N40, have contributed significantly to the understanding of Lyme disease pathogenesis and assessment of the virulence factors of B. burgdorferi [25-27]. B31 and N40 strains were isolated from Ixodes scapularis ticks from Shelter Island and Westchester county of New York, respectively, and both are highly infectious in the mouse model [2,28]. Indeed, N40 strain was selected for its high pathogenicity from a large number of isolates recovered from ticks by Durland Fish. By a thorough genetic analysis of various clones of N40 used in various laboratories, we have recently shown that the original culture was a mixed culture and different researchers isolated two different clones independently and retained the original name, N40, for both [29]. The clones designated as cN40 and the sequenced N40B are the derivatives of the same strain and N40 clone D10/E9 (N40D10/E9) and N40C appear to be derivatives of the second strain that is different from cN40/N40B. Comparative genomic analyses have indicated substantial genetic diversity between B31 and N40B [30]. For example, N40B possesses a smaller linear chromosome and contains fewer endogenous plasmids than the B31 strain [30]. To avoid further confusion, we will define specific N40 strains described above and in our recently published paper to determi (...truncated)