BvgAS is activated by growth at 37°C with low concentrations of nicotinic acid and sulfate (15). When B. bronchiseptica is cultured in SS liquid medium, type III secreted proteins are

detectable Erlotinib in the culture supernatant during the late logarithmic growth phase (6,16). However, the precise control mechanisms and environmental stimuli affecting expression of T3SS genes remain to be elucidated. Upon Bordetella colonization of the respiratory tract, the bacteria are exposed to severe environmental stress, especially iron-starvation. Host iron withholding systems such as lactoferrin serve to trap iron and withhold it from invading pathogens. As a result, the concentration of free iron in the extracellular tissue fluids of the host is approximately 10−18M (17). Thus, iron-starvation is one of the host INCB018424 innate defense systems, since a concentration of 4 × 10−7

to 4 × 10−6M of iron is required for bacterial growth (17). In order to counteract iron-starved conditions in the host, Bordetella has the uptake systems of the alcaligin siderophore, the enterobactin xenosiderophore, and heme for iron acquisition: these mechanisms allow bacterial survival in the host (18, 19). Thus, because stress conditions such as iron starvation determine the fate of invaded pathogens in the host, pathogens have evolved mechanisms for synergistic expression of virulence genes in response. Here, we demonstrate that iron starvation plays a critical role in T3SS expression in B. bronchiseptica. The wild-type strain used in this study was B. bronchiseptica S798 (6). An isogenic type III secretion mutant (T3SS−) was derived from the S798 strain (6). The Bordetella strains were cultured in SS liquid medium containing 0.5% casamino acids with a starting A600 of 0.2 under vigorous shaking at 37°C, and the inoculum prepared from fresh colonies grown on Bordet and Gengou agar, as described previously (20, 21, 22). Technical and

diphtheria toxin grades of casamino acids #223050 and #223120, respectively, were purchased from Difco Laboratories Atezolizumab purchase (Franklin Lakes, NJ, USA). The liquid cultivation period was 18 hr for the protein preparation/infection assay and 9 hr for mRNA preparation. Iron-depleted SS liquid medium was prepared by replacement of FeSO4 by MgSO4 at a final concentration of 36 μM, based on the recipe for the most commonly used SS medium (20, 21, 22). L2 (ATCC CCL-149) and HeLa (ATCC CCL-2) cells were maintained in F-12K (Invitrogen, Tokyo, Japan) and Eagle’s minimum essential medium (Sigma, St Louis, MO, USA), respectively, each supplemented with 10% FCS at 37°C in an atmosphere of 5% CO2. The anti-FhaB and anti-Prn antibodies used in this study have been described previously (23). The CyaA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).