The values represent the average copy number normalized per 100 copies of B. burgdorferi flaB transcripts. The cultivation of virulent B. burgdorferi in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats has been widely used a surrogate system for studying selected aspects of mammalian infection by B. burgdorferi [41]. However, although previous studies indicated that rpoS transcription was induced when B. burgdorferi was cultivated within rat DMCs [17], that check details approach represents a single temporal sampling that does not take

into account disseminatory events that occur URMC-099 manufacturer during natural mammalian infection. To better address this, we assessed rpoS transcription in mouse tissues at various times post-infection of mice via intradermal needle injection. rpoS transcripts were

readily detected in mouse tissues including skin, heart, and bladder at 7-, 14-, 21-, 28-, and 50-days post-infection (Figure 1B), suggesting that the RpoN-RpoS pathway is active during later disseminatory events of mammalian infection. To our knowledge, these are the first data indicating directly that activation of the RpoN-RpoS pathway is sustained throughout early and later phases of the mammalian infection process by B. burgdorferi. Expression of ospC, an RpoS-dependent gene, during tick and mouse infections Given the importance Thymidine kinase of OspC to the biology of B. burgdorferi infection [9, 13–15, 44, 45], and the

fact that ospC is a target of RpoS-mediated transcription [17, 19, 21, 46, 47], GSK458 ospC expression was assessed as a downstream marker of RpoN-RpoS activation. Transcription of ospC was barely detected in ticks during the acquisition phase (Figure 2A). However, in engorged nymphal ticks, ospC transcription was dramatically increased, which occurred in concert with rpoS transcription; at 24-, 48-, or 72-h after tick feeding, 35, 46 or 216 copies of ospC per 100 flaB transcripts, respectively, were detected (Figure 2A). These mRNA analyses are consistent with previous studies assessing OspC protein synthesis [7–9] and provide further evidence for the importance of OspC as an early factor critical for B. burgdorferi transmission from its tick vector to a mammalian host. Figure 2 qRT-PCR analysis of ospC transcription in ticks and in mouse tissues. A, flat (uninfected) larvae, fed larvae, intermolt larvae, and fed nymphs during transmission phase were collected at 24-, 48-, and 72-h post-feeding. TT: tick transmission. B, mouse tissues of skin (S) heart (H), and bladder (B) were collected at various numbers of days (inset) after infection. The values represent the average copy number normalized per 100 copies of B. burgdorferi flaB transcripts. We further examined ospC transcription within various mouse tissues.