Since BbCRASP-2 immunization did not influence spirochete infection, we created a BbCRASP-2-deficient B. burgdorferi to more directly assess the role of the gene product in B. burgdorferi survival and infectivity. An infectious isogenic mutant was created by replacing the BbCRASP-2 open reading frame with a kanamycin resistance Nutlin-3 cassette via homologous recombination. PCR analysis was performed to ensure that the antibiotic cassette was appropriately inserted into the intended chromosomal locus, and that the plasmid profile of the mutant was unchanged. Out of 4 transformed clones that grew in antibioticcontaining media, 2 clones contained the desired integration of the antibiotic cassette and retained the same set of plasmid as in the parental isolate. One of the mutant clones was chosen for further study. RT-PCR analysis showed that BbCRASP-2 mRNA was absent in the mutant, and that BbCRASP-2 mutagenesis did not impose polar effects on the transcription of surrounding genes, bbh05 and bbh07. The BbCRASP-2 mutant spirochetes displayed a similar protein profile to that of the wild type, except that the BbCRASP-2 mutant failed to produce BbCRASP-2 protein. To examine whether the lack of BbCRASP-2 influences B. burgdorferi infectivity in a mammalian host, C3H mice were infected with wild type or BbCRASP-2 mutant B. burgdorferi. Both the mutant and wild type spirochetes were readily cultured from ear and spleen tissues taken from mice 12 days after the inoculum. When nymphal ticks were allowed to feed on infected mice, BbCRASP-2 mutant B. burgdorferi were able to migrate into fed ticks at a similar level to the wild type spirochetes. Quantitative RT-PCR further showed that the BbCRASP-2 mutant established infection in mice in comparable levels to the parental isolate. No significant differences in the burdens of BbCRASP-2 mutant and wild type isolates were detected in murine skin, heart and joint samples isolated after 7, 12 and 18 days of infection. Development of swelling in the murine joints infected with either the BbCRASP-2 mutant or the wild type B. burgdorferi was also similar. Overall, these results suggest that BbCRASP-2 is not essential for establishment of B. burgdorferi infection in the mouse model of Lyme disease. B. burgdorferi express up to five BbCRASPs that are either structurally unique, such as BbCRASP-1 and -2, or closely Paclitaxel related, BbCRASP-3, -4 and -5. These BbCRASPs are differentially expressed and are postulated to confer defense against host-derived complements via specific interaction with FH family proteins. The precise role of individual BbCRASPs in the B. burgdorferi infection cycle, however, is currently unclear. BbCRASP-2 is specifically produced in the mammalian host including humans, and is immunogenic, and thus, is thought to be important in spirochete pathogenesis and may be useful in a future Lyme disease vaccine.