Shah, N.R., et al., Supplemental Data SUPPLEMENTAL TEXT. Cloning:

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1 Shah, N.R., et al., Supplemental Data SUPPLEMENTAL TEXT Cloning: pnmlgmab: the region containing lgma,lgmb, and the intergenic region upstream of lgma was amplified by PCR using genomic DNA of B. pertussis strain BP338 using primers BP398fw2 and BP399rev2. The PCR product was cloned into pbbr1mcs using XbaI and HindIII to generate pnmlgmab. pnmlgmcd: the region containing lgmc, lgmd, and the intergenic region upstream of lgmc was cloned into pbbr1mcs (1) using primers BP396fw1 and BP397rev1, and enzymes XbaI and HindIII to generate pnmlgmcd. pnmlgmabcd: the region containing lgma, lgmb, and the intergenic upstream of lgma was cloned from pnmlgmab into the vector pnmlgmcd using NheI and HindIII to generate pnmlgmabcd. pptaclgmabcd: the region containing the full lgm locus (lgma, lgmb, lgmc, lgmd) and the intergenic upstream region of lgma was cloned from pnmlgmabcd into pmmb67he using HindIII and KpnI to generate the vector pptaclgmabcd. pptaclgmab: the region containing lgma and lgmb was cloned from pnmlgmab into pmmb67he using HindIII and XbaI to generate the vector pptaclgmab. pbbr2pcpn: the Pcpn heat shock promoter was cut out of pbbrpcpnbrka using the KpnI sites, and cloned into the KpnI site of pbbr1mcs2 in the same direction as the kanamycin resistance cassette pbbr2lgma: the lgma gene was PCR amplified with primers lgmafw4 and lgmarev3, and the product was cloned into the ClaI and XbaI sites of pbbr2pcpn pbbr2lgmc: the lgmc gene was PCR amplified with primers lgmcfw3 and lgmcrev3. The PCR product was cloned into the and XbaI sites of pbbr2pcpn pptaclpxa338: lpxa from BP338 and the intergenic upstream region of lpxa were cut out of pbbrlpxa338 with HindIII and XbaI, and the ends were blunted with Klenow (New England Biolabs). pptaclgmabcd was cut with to cut out the LgmABCD locus, and the ends of the vector backbone were blunted with Klenow (New England Biolabs), into which the DNA fragment containing lpxa from BP338 and the intergenic upstream region of lpxa were cloned. A clone was selected in which the lpxa gene is in the forward direction in comparison to the pptac promoter, resulting in the vector pptaclpxa338. pptaclgmabcdlpxa338: lpxa from BP338 and the intergenic upstream region of lpxa were cut out of pbbrlpxa338 with HindIII and XbaI, and the ends were blunted with Klenow (New England Biolabs). pptaclgmabcd was cut with SapI, resulting in the 13.4 kb linear vector. The ends of this vector were blunted with Kelnow, and then pptaclgmabcd was ligated to the blunt-ended fragment of lpxa from BP338 and the intergenic upstream region. The resulting vector contains the LgmABCD locus under the control of the Ptac promoter, and the lpxa gene from BP338 under the control of its endogenous promoter. pss4245lgmadko: the lgma intergenic upstream region was amplified by PCR using genomic DNA of B. pertussis strain BP338 using primers lgmako1fw and lgmako1rev and the lgmd intergenic downstream region was amplified by PCR using genomic DNA of B. pertussis strain BP338 using primers lgmdko2fw and lgmdko2rev. These regions were cloned into the suicide vector pss4245 using,, and AvrII, such that the lgma intergenic upstream region is directly joined to the lgmd 1

2 intergenic downstream region via a restriction enzyme cut site sequence with two insertions (5 - TTTAATTAAG-3 ), resulting in pss4245lgmadko. pss4245lgmako: the lgma intergenic upstream and downstream regions were amplified by PCR using genomic DNA of B. pertussis strain BP338 using the primer sets (lgmako1fw and lgmako1rev) and (lgmako2fw and lgmako2rev), respectively. These regions were cloned into the suicide vector pss4245 using,, and AvrII, such that the lgma intergenic upstream region is directly joined to the lgma intergenic downstream region via a restriction enzyme cut site sequence. pss4245lgmbko: the lgmb intergenic upstream and downstream regions were amplified by PCR using genomic DNA of B. pertussis strain BP338 using the primer sets (lgmbko1fw and lgmbko1rev) and (lgmbko2fw and lgmbko2rev), respectively. These regions were cloned into the suicide vector pss4245 using,, and AvrII, such that the lgmb intergenic upstream region is directly joined to the lgmb intergenic downstream region via a restriction enzyme cut site sequence. pss4245lgmcko: the lgmc intergenic upstream and downstream regions were amplified by PCR using genomic DNA of B. pertussis strain BP338 using the primer sets (lgmcko1fw and lgmcko1rev) and (lgmcko2fw and lgmcko2rev), respectively. These regions were cloned into the suicide vector pss4245 using,, and AvrII, such that the lgmc intergenic upstream region is directly joined to the lgmc intergenic downstream region via a restriction enzyme cut site sequence. pss4245lgmdko: the lgmd intergenic upstream and downstream regions were amplified by PCR using genomic DNA of B. pertussis strain BP338 using the primer sets (lgmdko1fw and lgmdko1rev) and (lgmdko2fw and lgmdko2rev), respectively. These regions were cloned into the suicide vector pss4245 using,, and AvrII, such that the lgmd intergenic upstream region is directly joined to the lgmd intergenic downstream region via a restriction enzyme cut site sequence with one insertion (5 -CTTAATTAA-3 ), resulting in pss4245lgmdko BP338LgmABCDKO: the suicide vector pss4245lgmadko is used to generate a deletion mutant of the lgmabcd locus, using the previously described molecular cloning techniques (2). The resulting mutant, BP338SSLgmADKO, has the lgmabcd locus replaced by a restriction enzyme cut site sequence with two base pair insertions (5 -TTTAATTAAG-3 ). BP338LgmAKO: the suicide vector pss4245lgmako is used to generate a deletion mutant of the lgma gene, using the previously described molecular cloning techniques (3). The resulting mutant, BP338SSLgmAKO, has the lgma gene replaced by a restriction enzyme cut site sequence. BP338LgmBKO: the suicide vector pss4245lgmbko is used to generate a deletion mutant of the lgmb gene, using the previously described molecular cloning techniques (3). The resulting mutant, BP338SSLgmBKO, has the lgmb gene replaced by a restriction enzyme cut site sequence. BP338LgmCKO: the suicide vector pss4245lgmcko is used to generate a deletion mutant of the lgmc gene, using the previously described molecular cloning techniques (3). The resulting mutant, BP338SSLgmCKO, has the lgmc gene replaced by a restriction enzyme cut site sequence. BP338LgmDKO: the suicide vector pss4245lgmdko is used to generate a deletion mutant of the lgmd gene, using the previously described molecular cloning techniques (3). The resulting mutant, BP338SSLgmDKO, has the lgmd gene replaced by a restriction enzyme cut site sequence with one base pair insertion (5 - CTTAATTAA -3 ). 2

3 SUPPLEMENTAL REFERENCES Marr, N., Shah, N. R., Lee, R., Kim, E. J., and Fernandez, R. C. (211) Bordetella pertussis autotransporter Vag8 binds human C1 esterase inhibitor and confers serum resistance. PLoS One 6, e2585 Weiss, A. A., Hewlett, E. L., Myers, G. A., and Falkow, S. (1983) Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun 42, Inatsuka, C. S., Xu, Q., Vujkovic-Cvijin, I., Wong, S., Stibitz, S., Miller, J. F., and Cotter, P. A. (21) Pertactin is required for Bordetella species to resist neutrophil-mediated clearance. Infect Immun 78, SUPPLEMENTAL FIGURE LEGEND FIGURE S1. Structural analysis of lipid A with negative-ion MALDI mass spectra of the following B. pertussis strains: (A) BP338LgmAKO, (B) BP338LgmBKO, (C) BP338LgmCKO, (D) BP338LgmDKO, (E) BP338LgmAKO + pbbr2lgma (complementation of BP338 lgma mutant), (F) BP338LgmBKO + pptaclgmab (complementation of BP338 lgmb mutant), (G) BP338LgmCKO + pbbr2lgmc (complementation of BP338 lgmc mutant), (H) BP338LgmABCDKO + pptaclgmabcd (complementation of BP338 full Lgm locus mutant in trans with the full Lgm locus, lgmabcd), (I) BP338LgmABCDKO + pptaclgmabc (complementation of BP338 full Lgm locus mutant in trans with lgmabc), (J) pmmb67he ( wild type complemented with an empty vector, a control for the pptac vectors, as seen in Fig. 4 B, C, D). For (A-I), peaks at m/z 1559 represent penta-acyl lipid A that lack modification, peaks at m/z172 represent penta-acyl lipid A with one modification at either phosphate group, and peaks at m/z 1881 represent penta-acyl lipid A with two modifications, one at each phosphate group. For (J), peaks at m/z 152 and 153 represent penta-acyl lipid A with C1OH or C12OH at the C3 position, respectively. FIGURE S2. TLR4 activity of purified LOS from strains as measured with the HEK-Blue NFκB TLR4 activity assay. Null2 all, stimulation of HEK-Blue Null2 cell line that lacks TLR4 expression with all LPS variants; Blank, media only with no HEK-Blue cells; Unstim, HEK-Blue htlr4 cells stimulated with media only; pptaclpxa338, complemented with lpxa from BP338; pptaclgmabcd, complemented with Lgm locus from BP338; pptaclgmabcdlpxa, complemented with both lpxa and Lgm locus from BP338. Stimulation with LOS at concentrations: 1 ng/ml (green), or ng/ml (red). Relative differences in NFκB activity levels upon stimulation with ng/ml of LOS from the indicated strains were similar to what was seen upon stimulation with 1 ng/ml of LOS (Fig. 6). At 1 ng/ml of purified LOS, there was no significant difference in the relative responses to the different strains. Graph shows the results of one representative experiment of three, n = 5. P-values: <.1 (**), <.1 (***). 3

4 SUPPLEMENTAL TABLE Table S1. Primers Primer Restriction enzyme site Sequence (5 to3 ) BP399fw1 ATCTGTCTGGACGCCGACATGC BP399rev1 CAGGTAACCGCCGTAGGACAGC BP399rev2 CCCCAAGCTTGTCCCAGGGATAGGCGATCAGG BP398fw1 TTCTTCGTCCACCAGCATTTCG BP398rev1 AGCTGCAGGTCGAACGGATAGG BP398fw2 CGCTCTAGAGGCTGGCCTTCCTGACATTCG BP397rev1 CCCCAAGCTTCAAGGGGCGTGTCTGTGACG BP397-RTfw CGGAACATTCCGGACCTTACCC BP397-RTrev CCACCAGCACGTGCATCAGC BP396fw1 CGCTCTAGATCAAGCGGCTGGTGAAGATCG BP396-RTfw GCGCGCAGCTTGTCATAGGC BP396-RTrev CAAACCCCCAAAACCATCAAGG BP338lpxAfw1 GGAATTCGATCCGTAGCCTGGAAGAG lgmako1fw GGAATTCTGGTAGCCGTGCCGCAGCC lgmako1rev CCTTAATTAAACCACGGCGAAATTGACGGG lgmako2fw CCTTAATTAATGACTCTCGCTACCCGATCC lgmako2rev CCCCTAGGCAGCGTGGCCAGCACCAG lgmafw4 CCATCGATTTCGCCGTGGTGTGTTCATG lgmarev3 GCTCTAGATCATTGGGAACGCGCCTTG lgmbko1fw CGAATTCGCGACTTCCGCCTGATGGAC lgmbko1rev CCTTAATTAATCATTGGGAACGCGCCTTGGC lgmbko2fw GCTTAATTAACGACGAATGTCAGGAAGGCCA lgmbko2rev CCCCTAGGGACCGCGCTCAGGCGTC lgmcko1fw GGAATTCGCCGATGTGGGTGGTCGAC lgmcko1rev CCTTAATTAATCAGACTCACTTTCCGCCAC lgmcko2fw CCTTAATTAAAATGAGTTCTTCCTCGAGACAAAC lgmcko2rev CCCCTAGGTCCGTGGCCGACTTCGTACG lgmcfw3 GGAATTCCATGACCAGTGAACGATACGA lgmcrev3 GCTCTAGATCATAAACGGCTTGCCAG lgmdko1fw GGAATTCAGCGCATCTGGCTGCGG lgmdko1rev CCTTAATTAATCATAAACGGCTTGCCAGGC lgmdko2fw CCTTAATTAAGGCCGCTCAGGTACCGGGC lgmdko2rev CCCCTAGGACTGCCCTCGGAGCAAAGCG The restriction enzyme cut site present in each primer is underlined in the sequence 4 HindIII XbaI HindIII XbaI AvrII ClaI XbaI AvrII AvrII XbaI AvrII

5 BP338LgmAKO A B X C BP338LgmCKO D BP338LgmDKO Intensity (arb. u.) BP338LgmBKO Mass (m/z) Figure S1 18 2

6 BP338LgmAKO+pBBR2LgmA E BP338LgmBKO+pPtacLgmAB F BP338LgmCKO+pBBR2LgmC G Intensity (arb. u.) X BP338LgmABCDKO+pPtacLgmABCD H BP338LgmABCDKO+pPtacLgmABC I Figure S1, cont'd Mass ( m/z )

7 Intensity (arb. u.) C12OH J pmmb67he C1OH X Mass (m/z) Figure S1, cont'd 18 2

8 *** *** *** *** ** Figure S2