Studies in B. burgdorferi demonstrate that OspA and OspB mediate spirochete association with the tick midgut epithelium shortly after ingestion [3–5], a process that would presumably be facilitated by a chitinase activity. A similar mechanism for vector colonization has been investigated in other organisms that cause vector-borne disease. It has been demonstrated in Leishmania [20] and Plasmodium [21, 22] that chitinases and N-acetylglucosaminidases

play a role #BLZ945 price randurls[1|1|,|CHEM1|]# in weakening the peritrophic membrane, thereby allowing invasion of the midgut epithelium of the sandfly and mosquito, respectively. Inspection of the B. burgdorferi genome reveals both enzymes and transporters that may be involved in chitin degradation. There are two genes predicted to be involved in the cleavage of β-(1,4) glycosidic bonds, a putative

β-N-acetylhexosaminidase (bb0002) and a putative β-glucosidase (bb0620). In addition, previous reports have characterized the chitobiose transport system in B. burgdorferi, which is encoded on circular plasmid 26 (bbb04, bbb05 and bbb06) [14, 15, 17]. It is possible that this transport system plays a role in the utilization of chitin breakdown products (i.e. chitobiose), a mechanism that has been investigated in other chitin-degrading microorganisms [23, 24]. As described above, B. burgdorferi cannot generate GlcNAc de novo and must import this essential sugar from the surrounding environment. Therefore, during in vitro propagation the addition of free GlcNAc is necessary for PF477736 cells to reach optimal cell densities in a single exponential phase. In the absence of free GlcNAc, B. burgdorferi exhibits a unique biphasic growth pattern. In the first exponential phase cells utilize the residual GlcNAc and chitobiose present in complex medium components and grow to

approximately 2.0 × 106 cells ml-1 [14, 17]. Edoxaban Cells then become starved for GlcNAc and exhibit a death phase in which cell numbers decrease to 1.0 × 105 cells ml-1. By 120 hours cells begin to grow in a second exponential phase and reach cell densities greater than 1.0 × 107 cells ml-1. While the source of GlcNAc in the second exponential phase remains unknown, it is possible that sequestered forms of this sugar such as chitin or glycoproteins present in complex medium components play a role. The goals of this study were to determine if B. burgdorferi could utilize chitin as a source of GlcNAc and to identify genes important in the process. Results Chitinase activity in rabbit serum Previous reports have described a chitinase activity in mammalian tissues and serum [25–28]. In order to investigate chitin utilization by B. burgdorferi, we first determined if there was an inherent chitinase activity in the growth medium (BSK-II) that would interfere with subsequent growth analyses of B. burgdorferi in the presence of chitin.