Analysis of Mechanisms Associated with Loss of Infectivity of Clonal Populations of Borrelia burgdorferi B31MI

Abstract

Numerous studies have provided suggestive evidence that the loss of plasmids correlates with the loss of infectivity of the Lyme disease spirochetes. In this study we have further investigated this correlation. Clonal populations were obtained from the skin of a mouse infected for 3 months with a clonal population of Borrelia burgdorferi B31MI. The complete plasmid compositions of these populations were determined using a combination of PCR and Southern hybridization. The infectivities of clones differing in plasmid composition were tested using the C3H-HeJ murine model for Lyme disease. While several clones were found to be noninfectious, a correlation between the loss of a specific plasmid and loss of infectivity in the clones analyzed in this report was not observed. While it is clear from recent studies that the loss of some specific plasmids results in attenuated virulence, this study demonstrates that additional mechanisms also contribute to the loss of infectivity.Infection with pathogenic species of the Borrelia burgdorferi sensu lato complex can lead to Lyme disease, an infection characterized by highly variable multisystem clinical manifestations (30, 31). Early studies suggested that plasmid-encoded proteins play an important role in Borrelia pathogenesis (26,28), and in recent years it has been demonstrated that they also play an important role in immune evasion (37,43). In addition, several plasmid-borne genes have been demonstrated to be up-regulated during infection, supporting a functional role for the proteins in the mammalian environment (1,11,39). The ability to define a correlation between specific plasmids and infectivity has until recently been complicated by the inherent instability of the Borrelia plasmids and the variation in plasmid content among isolates (3,9,17,18,20,25,28,29,40,41). Prior to the determination of the complete genome sequence and plasmid content of B. burgdorferi, these features made it difficult to interpret and compare much of the earlier data. Determination of the genome sequence of B. burgdorferi B31MI and other analyses revealed the existence of both linear and circular comigrating plasmids of between 24 and 56 kb that could not be distinguished by agarose gel electrophoresis (5, 10, 15, 35). There are also several distinct yet closely related circular plasmids (designated cp) of approximately 32 kb (cp32) (33, 35) and multiple linear plasmids (designated lp) of approximately 28 kb (lp28s). The cp32s have extended regions of homology, while the lp28s have significantly less identity. In addition to comigrating plasmids, the genome also contains an extraordinary number of plasmid-carried paralogous gene families (n ϭ 175). This genetic redundancy, which could allow for functional complementation of plasmid-encoded proteins, coupled with the inherent instability of the Borrelia plasmids has made it difficult to pinpoint one or more specific plasmids as essential for infectivity (4,9,17,28,37,38,43). However, two recent independent studies by Labandeira-Rey and Skare...

Role of the BBA64 Locus of Borrelia burgdorferi in Early Stages of Infectivity in a Murine Model of Lyme Disease

“…The process of generating mts may inadvertently result in selection of mutations in distal genes or loss of plasmids with significant effects on the infectivity of B. burgdorferi (36,41,51,64). There were no overt differences in the protein profile and the level of OspC was significantly higher under mammalian host-specific conditions in all three strains, indicating that both the regulatory mechanisms and the expression of other critical determinants of virulence were similar and intact in the mt and control strains (1,58,63,67).…”

Inactivation of bb0184 , Which Encodes Carbon Storage Regulator A, Represses the Infectivity of Borrelia burgdorferi

“…After several attempts, we found that the obtained antibiotic-resistant clones lost either lp28-1 or lp25, two linear plasmids that are essential for the infectivity of B. burgdorferi (35,43). To solve this problem, the shuttle vector pBBE22G was used to construct the plasmid CsrA/ pBBE22G (Fig.…”

Evidence for the Contribution of Point Mutations tovlsEVariation and for Apparent Constraints on the Net Accumulation of Sequence Changes invlsEduring Infection with Lyme Disease Spirochetes

“…While the vls system has been extensively characterized in B31-5A3 (19)(20)(21), little is know about the kinetics of vlsE variation in other Lyme disease isolates or clones. We selected B31MIpc for these analyses since the majority of its genome sequence, including its vls cassettes, has been determined (5) and its plasmid content has been verified (13). An essential starting point for these analyses was to verify the presence, map location, and sequence of vlsE in B31MIpc.…”