Evidence for the contribution of point mutations to vlsE variation and forapparent constraints on the net - Accumulation of sequence changes in vlsEduring infection with Lyme disease spirochetes

In the Lyme disease spirochetes, both the ospE and vlsE gene families have been demonstrated to undergo sequence variation during infection. To furthe r investigate the mechanisms associated with the generation of vls variatio n, single-nucleotide polymorphism and subsequent DNA sequence analyses were performed on the vlsE gene and its paralog, BBJ51, a related gene with a f rameshift mutation. These analyses focused on a series of postinfection clo nal populations obtained from mice infected with Borrelia burgdorferi B31MI pc or its clonal derivative, B31MIc53. vlsE, but not BBJ51, was found to un dergo sequence changes during infection. Consistent with that reported prev iously (J.-R. Zhang et al., Cell 89:275-285, 1997) many of the sequence cha nges appear to have arisen through gene conversion events and to be localiz ed to the variable regions of vlsE. However, analysis of the vlsE nucleotid e sequences revealed that some sequence changes were the result of point mu tations, as these changes did not have potential contributing sources in th e vls cassettes. To determine if sequence changes accumulate in vlsE over l ong-term infection, the vlsE genes of clonal populations recovered after 7 months of infection in mice were analyzed. While new sequence changes devel oped, a significant number of these changes resulted in the restoration of the vlsE sequence of the original infecting clone. In addition, we noted th at some positions within the variable regions (VR) are stable even though t he cassettes possess residues that could contribute to sequence variation t hrough gene conversion. These analyses suggest that the total number of ami no acid sequence changes that can be maintained by VlsE levels off during i nfection. In summary, in this report we demonstrate that the development of point mutations serves as a second mechanism by which vlsE sequence variat ion can be generated and that the capacity for vlsE variation, while still significant, is less than previously postulated.