generate plasmid pIRI-2, sequenced to ensure the maintenance of the reading frame, and subcloned into the BamHI and the XhoI sites of the suicide plasmid pJQ200KS. The mutator plasmid (pLRI-7) was introduced in BAB-parental by conjugation [78]. Integration of the suicide vector was selected by Nal and Gm resistance, and the excision (generating the mutant strain by allelic exchange) was selected by Nal and sucrose resistance and Gm sensitivity. The resulting colonies were screened by PCR with primers olsB-F1 and olsB-R4 which amplify a fragment of 564 bp in the mutant and a fragment of 1134 bp in the parental strain. The mutation resulted in the loss of the consensus amino acids responsible for the enzymatic activity. The BABDolsA mutant was constructed using primers olsA-F1 (59- GATTGCGCAGGATACCATCT -39) and olsA-R2 (59- AACAATGCGGTGGAAGAAAT-39) to amplify a 498- bp fragment including codons 1 to 47 of the olsA ORF, as well as 357-bp upstream of the olsA start codon and primers olsA-F3 (59ATTTCTTCCACCGCATTGTTACGATGGAAAATCGG- GTGAG- 39) and olsA-R4 (59-CAGCGCGGAATAGAGTTTTT- 39) to amplify a 372-bp including codons 246 to 268 of the olsA ORF and 303 bp downstream of the olsA stop codon. Both fragments were ligated by overlapping PCR using primers olsA-F1 and olsA-R4 and the fragment obtained, containing the deletion allele, was cloned into pCR2.1 to generate pYLI-2, sequenced to confirm that the reading frame had been maintained, and subcloned in pJQ200KS to produce the mutator plasmid pYLI- 3. This plasmid was introduced in BAB-parental and the deletion mutant generated by allelic exchange was selected by Nal and sucrose resistance and Gm sensitivity and by PCR using oligonucleotides olsA-F1 and olsA-R4 which amplify a fragment of 870 bp in the deletion strain and a fragment of 1464 bp in the parental strain. The mutation generated results in the loss of 73.8% of the olsA ORF. For complementation, plasmids carrying olsB and olsA were constructed using the Gateway cloning Technology (Invitrogen). Gene olsB was amplified from BAB-parental using primers olsB- F13 (59 GGGGACAAGTTTGTACAAAAAAGCAGGCTT- CATGACAGCACTGCTTGGAATGG 39) and gene olsB- R14 (59 GGGGACCACTTTGTACAAGAAAGCTGGGTC CTAG- ACAAAGCGGTTTGCTTC 39), that contain the attB sequences (underlined), and cloned into vector pDONR221 (Invitrogen) to generate pLPI-5. The ORF was subsequently cloned in pRH001 [79], able to multiply in Brucella, to produce the complementation plasmid pLPI-6. Since the sequence of olsA from B. abortus and B. melitensis is 99% identical, the clone carrying olsA was extracted from the B. melitensis ORFEOMA [80] and the ORF subcloned into plasmid pRH001 [81] to produce plasmid pYLI-1. To complement the olsB mutation, plasmid pLPI-6 was introduced into the BABDolsB mutant by mating with E. coli S17 lpir and the conjugants harboring this plasmid (designated as BABDolsB pLPI- 6) were selected by plating the mating mixture onto tryptic soy agar-Nal-Km plates. The olsA mutation was complemented following the same protocol by introducing plasmid pYLI-1 into the BABDolsA mutant. Lipid analysis Total lipids were extracted as described by Bligh and Dyer [82], and analyzed on silica gel 60 high-performance thin layer chromatography plates (Merck Chemicals), the plates were pre- washed by solvent migration with chloroform–methanol-water (140:60:10, vol/vol), dried thoroughly. Then samples were applied and chromatography performed in the same mixture of solvents [83]. Plates were developed with 0.2% ninhydrin in acetone and heating at 120uC for 5 min or by charring with 15% sulfuric acid Brucella Ornithine Lipids in ethanol at 180uC. L-b,c-dipalmitoyl-a cephalin (Sigma-Aldrich) was used as a standard. Sensitivity to non immune serum and complement consumption Exponentially growing bacteria were adjusted to 104 CFU/ml and mixed with fresh sheep normal serum (45 ml of cells plus 90 ml of serum per well) in microtiter type plates in duplicate. After incubation for 90 min at 37uC with gentle stirring, brain heart infusion broth (200 ml/well) was added, mixed and 100 ml aliquots plated out by triplicate. The results were expressed as the % survival with respect to the CFU in the inocula. Complement consumption was estimated as the reduction of the hemolytic activity of rabbit serum complement incubated with live bacteria, and S. typhimurium SL1344 was used as a positive control [1]. Infections and bacterial counts in cells Bone marrow cells were isolated from femurs of 6–10-week-old C57Bl/6 female mice and differentiated into macrophages [84]. Infections were performed at a multiplicity of infection of 50:1 by centrifuging bacteria onto macrophages at 400 g for 10 min at 4uC, followed by a15 min incubation at 37uC under a 5% CO2 atmosphere. Macrophages were extensively washed with Dulbec- cos’s modified Eagle’s medium to remove extracellular bacteria and incubated for an additional 90 min in medium supplemented with 50 mg/ml gentamicin to kill extracellular bacteria. Thereafter, the antibiotic concentration was decreased to 10 mg/ml. At each time point, samples were washed three times with 100 mM PBS before processing. Alternatively, murine RAW 264.7 macrophages (Raw 264.7; American Type Culture Collection No. TIB-71) or Hela cells (American Type Culture Collection No. CCL-2) were used in a similar protocol. To monitor Brucella intracellular survival, infected cells were lysed with 0.1% (vol/vol) Triton X-100 in H2O after PBS washing and serial dilutions of lysates were rapidly plated onto TSB agar plates to enumerate CFU. The attenuated B. abortus virB mutant (Table S1) was used as a control. OL stimulation of macrophages For macrophage stimulation, OL were included in liposomes. Total free-lipid extracts of BAB-parental, BABDolsB or a 1:1 mixture of B. pertussis and BABDolsB free-lipids were evaporated under a nitrogen stream, resuspended thoroughly in 250 ml of 10mM HEPES to a final concentration of 5 mg/ml, and the composition verified by thin-layer chromatography. Phase contrast microscopy and fluorescein entrapment controls demonstrated that Brucella total lipids formed liposomes that were stable for at least 24 h without addition of exogenous lipids. The murine RAW 264.7 macrophages used in these experiments were grown at 37uC under 5% CO2 in Dulbecco’s medium supplemented with 10% fetal bovine serum, 2.5% sodium bicarbonate, 1% glutamine (all from Gibco), penicillin (100 units/ml) and streptomycin (100 mg/ ml). Macrophages (56105 cells) were treated with 20 mg/mL or 100 mg/mL of liposomes of BAB-parental, BABDolsB or B. pertussis-BABDolsB in 10% fetal calf serum-Dulbecco’s Modified Eagle’s Medium. After 90 min of incubation, fresh 10% fetal calf serum- Dulbeccos’s modified Eagle’s medium was added to obtain a final concentration of 10 mg/ml and 50 mg/ml of liposomes, and the amount of TNF-a and IL-6 in the supernatants was assessed by ELISA (eBioscience) after 6 and 24 h. Proinflammatory responses in mice Swiss CD1 mice of 18 to 20 g were used. Fibrin D- dimers were determined from the plasma of mice 24 h after intraperitoneal PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16030