Methods Bacterial strains and cultures Y. pestis CO92 and Y. pestis CO92 Δcaf1ΔpsaA were transformed with pGEN-luxCDABE [24]. This plasmid contains the Hok/Sok toxin/antitoxin system enabling plasmid maintenance in vivo without antibiotic selection. Throughout this document we referred to Y. pestis CO92 transformed with the pGEN-luxCDABE plasmid as Yplux +, to Y. pestis CO92 Δcaf1ΔpsaA transformed with the same plasmid as YpΔcaf1ΔpsaAlux + or simply as “double mutant” and to FK228 the pGEN-luxCDABE plasmid itself as pGEN-lux. Bacteria transformed

with pGEN-lux were cultured in the presence of carbenicillin at 100 μg/mL, unless BHI alone is stated as growth medium. Bacteria were plated on brain heart infusion (BHI) agar (BD Biosciences, Bedford, MA) plates and incubated for 48 h at 26°C. For intranasal

inoculations, liquid cultures SN-38 cost were incubated at 37°C in the presence of 2.5 mM CaCl2 as previously described [29]. For subcutaneous and intradermal inoculations, liquid cultures were incubated at 26°C for 15 h. All strains (Yplux +, YpΔcaf1ΔpsaAlux + and Y. pestis lacking pGEN-lux) showed comparable optical density (OD600) values after culturing in liquid broth. To obtain the final inocula for all infections, liquid cultures were serial diluted in phosphate buffered saline (PBS). All procedures involving Y. pestis were conducted under strict biosafety level three conditions. Animal infections and tissues Five-to-ten-week old female C57BL/6J or B6(Cg)-Tyrc-2J/J mice (Jackson Laboratory, Bar Harbor, ME) were subjected to subcutaneous (SC), intranasal (IN) or intradermal (ID) inoculation after providing anesthesia (2% isoflurane for SC and

ketamine/xylazine for IN and ID). For SC inoculations, a volume of 100 μL was injected in the subcutaneous space at an Sapitinib in vivo anterior cervical site. The ear pinna was injected with a volume of 10 μL for ID inoculations. A volume of 20 μL was delivered into the left nostril of the animal for IN inoculations. The inoculum for the SC and ID inoculations was ~200 CFU, and ~104 CFU for the IN inoculation. For the determination Cepharanthine of plasmid stability and strain characterization experiments, superficial cervical lymph nodes, spleens and lungs were removed from SC-infected mice after sacrificing the animals by injection of sodium pentobarbital. Plasmid stability was assessed by comparing bacterial counts after plating on BHI alone and BHI with carbenicillin. Strain characterization was determined by comparing bacterial counts of Yplux + against Y. pestis lacking the plasmid. All procedures involving animals were approved by the University of North Carolina and Duke University Animal Care and Use Committees, protocols 11–128 and A185-11-07, respectively.