Strains and culture conditions. H. influenzae
Rd, a capsule-deficient serotype d derivative (71
), and a virulent streptomycin-resistant derivative of H. influenzae
type b strain Eagan (Hib) (5
) were grown in brain heart infusion broth (BHI) supplemented with 10 μg/ml hemin and 10 μg/ml NAD (sBHI), in MIc, a low-nutrient medium capable of supporting growth of H. influenzae
), or on sBHI agar plates at 35°C. Development of competence for transformation of H. influenzae
was accomplished as previously described (7
). For selection of Rd- and Hib-derived strains, antibiotics were used at the following concentrations: 8 μg/ml tetracycline, 20 μg/ml kanamycin, 10 μg/ml gentamicin, and 100 μg/ml streptomycin.
dsbA strain construction.
Plasmids and PCR products were constructed using standard molecular biology techniques (6
). For complementation of mutants, DNA fragments were amplified by PCR and cloned between adjacent SapI restriction sites of the chromosomal delivery vector pXT10, which does not replicate in H. influenzae
). The pXT10-based plasmids contained upstream (xylF
) and downstream (xylB
) homologous regions flanking the SapI cloning sites that allowed precise fusion of genes of interest to the xylose-inducible xylA
promoter, as previously described (71
). Recombination at the xylose catabolic locus replaced the endogenous xylA
gene with the cloned fragment and the tetAR
tetracycline resistance cassette. Plasmids were linearized by digestion with PciI and SacI, and tetracycline-resistant (Tetr
) recombinants were selected on sBHI agar plates. Double crossovers within xylF
were confirmed by performing PCR with primers specific for sequences outside the inserted recombinant region.
To generate a dsbA mutant and a complemented strain of H. influenzae which requires DsbA for natural transformation, we first generated a strain containing an inducible copy of dsbA and sequentially introduced the dsbA deletion and the complementation construct or the “empty vector” construct into this background. Initially, an additional copy of dsbA under control of the xylose-inducible promoter of xylA was introduced into H. influenzae Rd to create strain RX. The coding sequence of DsbA lacking the translational termination codon was amplified by PCR with primers F-NTdsb (5′-AAAGATCTGCTCTTCAATGAAAAAAGTATTACTTGC-3′) and 3dsbAHA (5′-AAAGATCTGCTCTTCGTAATGCATAATCTGGCACATCATATGGATATTTTTGCAATAAACCTTTTACGGTT-3′), which introduced SapI sites in the termini of the fragment. The resulting fragment was cloned into pXT10 that had been digested previously with SapI. The resulting plasmid, pXyldsbA1.1, was linearized and used to transform H. influenzae to tetracycline resistance to create strain RX.
Next, the native copy of dsbA was deleted from RX by replacement with the aacC1 gentamicin resistance gene to create strain RdsbAX by PCR “stitching” as follows. Overlapping PCR fragments generated with primers representing the 951-bp region immediately 5′ of the dsbA translational start codon (primers 5844H [5′-TTTAAGCTTTTAGATGACTGTTTTCTTTAAATC-3′] and 3Dsbout [5′-TTCTTTCCTCTTATTTAATGATACCGCGAG-3′]), the 569-bp aacC1 gene encoding gentamicin resistance (primers 5GentD [5′-TAAATAAGAGGAAAGAAATGTTACGCAGCAGCAACGATGTT-3′] and 3GentD [5′-CATTAAACCAATTTTTCGTTAGGTGGCGGTACTTGGGTCGAT-3′]), and the 1,641-bp 3′ region starting at the dsbA termination codon (primers 5Dsbout [5′-CGAAAAATTGGTTTAATGCCAGCCC-3′] and 3848H [5′-TTTAAGCTTCTACTTGCGAATGAGCCATAGGC-3′]) were combined by overlap extension PCR with primers 5844H and 3848H to precisely replace the dsbA coding sequence with the coding sequence of aacC1. The resulting 3,126-bp DNA fragment was used to transform strain RX, and gentamicin-resistant (Gmr) recombinants were isolated to create strain RdsbAX, which contained a single copy of dsbA under control of the xylose-inducible xylA promoter.
To complement the dsbA knockout with a wild-type copy of dsbA under control of its own promoter, overlap extension PCR was performed as follows. Primers pXT10thyA-F (5′-AGGGCTTGAATCGCACCTCCA-3′) and 3dsbkan1 (5′-CATCAGAGATTTTGAGACACGGGCCTCTTATTTTTGCAATAAACCTTTTACGGT-3′) were used in PCR to amplify a 1,983-bp fragment containing dsbA from a pXT10-based plasmid carrying dsbA coding sequences, pDsbA1.2. A 2,716-bp PCR product was amplified from a kanamycin-marked derivative of pXT10 with primers 5pkan1 (5′-GAGGCCCGTGTCTCAAAATCTCTGATG-3′) and 3revRfaD1 (5′-AACAGGCTACGATAAACCATTCAAAACAGT-3′). The 1,983- and 2,716-bp fragments were joined via the 27 bp of overlapping sequence by PCR performed with primers pXT10thyA-F and 3revRfaD1, the resultant 4,672-bp PCR product was transformed into strain RdsbAX (grown in the presence of 1 mM d-xylose to induce expression of dsbA), and kanamycin-resistant (Kmr) transformants were isolated to create strain RdsbAC.
To control for effects of modification of the xyl locus, a dsbA mutant containing the integrated “empty vector” sequences was generated by transforming RdsbAX grown in the presence of 1 mM d-xylose with a 4,334-bp PCR product having a precise deletion of the dsbA coding sequences of the 4,672-bp construct described above in RdsbAX, except that primers 3xylF1 (5′-ACGTTTATCAACAGCGATAGGATCAAGT-3′) and 3pDsbAsapKan (5′-CATCAGAGATTTTGAGACACGGGCCTCTTACGAAGAGCGGCGCGCCGCTCTTCCCATTTCTTTCCTCTTATTTAATGATACCGCGA-3′) were used instead of primers pXT10thyA-F and 3dsbAkan. Selection for Kmr transformants resulted in isolation of strain RdsbAV. To construct a strain that contained the “empty vector” in a wild-type background, the same 4,334-bp PCR product was transformed into H. influenzae Rd, and Kmr transformants were isolated to create strain RXV.
Similarly, the same set of constructs was used to generate the dsbA mutant HdsbAV, a vector-only strain (HXV), and a complemented strain (HdsbAC) in the H. influenzae type b strain Eagan background. The wild-type and dsbA mutant phenotypes of all strains were verified using a dithiothreitol (DTT) sensitivity assay (described below), and all mutations were confirmed by sequence analysis of the recombinant loci.
HbpA strain construction.
hbpA mutant strain RhbpA was constructed by replacement of the coding sequence of hbpA with the kanamycin resistance gene, aphI. The exchange fragment was synthesized by overlap extension PCR between three regions: a 1,083-bp PCR product containing the 5′ flanking region of hbpA generated using primers 5hbp1 (5′-AGTCATTCACGCCAGTTGGCACTGGAT-3′) and 3hbp1 (5′-TTCCCGTTGAATATGGCTCATACCTCAATGTTAGGCAGGGAATGCCCTA-3′), an 816-bp PCR product containing the coding region for the kanamycin resistance gene generated with primers 5kan1.1 (5′-ATGAGCCATATTCAACGGGAA-3′) and 3kan1.1 (5′-TTAGAAAAACTCATCGAGCATCAAATG-3′), and a 1,020-bp PCR product containing the 3′ flanking region of hbpA generated with primers 5hbp3 (5′-CATTTGATGCTCGATGAGTTTTTCTAATTCATATTGATTTACTTATTTTAAGCCCT-3′) and 3hbp3 (5′-CAAAAGGGGTGAGTATAAATTTACACTCAA-3′). The 1,083-, 1,020, and 816-bp fragments were joined in a PCR via their complementary ends using primers 5hbp1 and 3hbp3. The resulting 2,871-bp fragment was introduced into H. influenzae Rd, and Kmr transformants were selected on sBHI containing kanamycin to create strain RhbpA. To construct an hbpA knockout mutant carrying the integrated empty exchange vector, strain RhbpA was transformed with linearized vector pXT10, and Tetr transformants were isolated to create strain RhbpAV.
To complement the hbpA mutation with a copy of hbpA expressed from the hbpA promoter, a 1,842-bp fragment containing the hbpA coding region and including 142 bp upstream of hbpA was amplified from Rd using primers 5hbpha (5′-AAAGCTCTTCAATGATTAATTTGTTATAATCCATAGA-3′) and 3hbpha (5′-TTTGCTCTTCTTTATGCATAATCTGGCACATCATATGGATATTTACCATCAACACTCACACCATA-3′). This set of primers also added a C-terminal hemagglutinin (HA) epitope tag to hbpA. The PCR product was cloned between the two SapI sites of pXT10 to generate plasmid pXhbp1.5, which was then introduced into strain RhbpA with selection for Tetr to create strain RhbpAC. To introduce a nonpolar, in-frame deletion of dsbA into strain RhbpAC, this strain was transformed with the 3,126-bp dsbA replacement fragment described above, and Gmr transformants were selected to create strain RhbpACΔdsbA.
Strain RdV carrying pXT10 “empty vector” sequences in the xyl
locus and strain RdlacZ (H. influenzae
Rd carrying lacZ
at the xyl
locus) were constructed as previously described (72
). Strain RdgalU was constructed by replacement of galU
with the aphI
cassette. For all mutant strains, replacement of endogenous loci by double-crossover homologous recombination with mutant constructs was confirmed by PCR performed with primers specific for sequences flanking the inserted recombinant region.
DTT sensitivity assay.
To evaluate sensitivity to DTT, strains were inoculated in triplicate using inocula from overnight cultures into 25 ml of sBHI in 50-ml Erlenmeyer flasks to obtain an optical density at 600 nm (OD600) of 0.01 and incubated at 35°C with shaking at 250 rpm. When cultures reached the log phase, they were diluted in sBHI to obtain an OD600 of 0.02, and 100 μl was transferred to a 96-well flat-bottom dish. Each well in the dish was then treated with 100 μl of sBHI containing 10 mM DTT at a final concentration of 5 mM or with sBHI alone (control wells). The dish was then incubated at 35°C for 16 h in a Versamax microplate reader (Molecular Devices, Sunnyvale, CA) set to read the absorbance at 600 nm every 10 min. Sensitivity was assessed using the relative OD600 values at the end of the incubation period.
Hydrogen peroxide sensitivity.
To determine the sensitivity of the dsbA deletion mutant to H2O2, strains Rd, RXV, RdsbAV, and RdsbAC were inoculated using inocula from overnight cultures in triplicate into 25 ml of sBHI in 50-ml Erlenmeyer flasks or into 5 ml of sBHI in culture tubes to obtain an OD600 of 0.01. The resulting cultures were incubated aerobically at 35°C with shaking at 250 rpm (flasks) or in an anaerobic chamber (culture tubes) with BBL GasPak Plus generators (Becton, Dickinson and Company, Sparks, MD). When cultures reached the log phase, they were diluted in sBHI to obtain an OD600 of 0.02, and 100 μl of each culture was seeded into a 96-well flat-bottom dish. Hydrogen peroxide (Sigma-Aldrich, St. Louis, MO) diluted in 100 μl of sBHI was then added to cultures grown in 25-ml flasks at final concentrations of 0, 62.5, 125, and 250 μM in sBHI and to anaerobically grown cultures at final concentrations of 0, 62.5, 125, and 500 μM. The dishes were then incubated at 35°C for 16 h in a microplate reader, and the absorbance at 600 nm was determined every 10 min to evaluate the growth rates and final culture densities.
Growth of dsbA strains.
To determine the growth rates in rich media and in defined media, strains were inoculated in triplicate to obtain an OD600 of 0.01 by using inocula from standing overnight cultures into 25-ml Erlenmeyer flasks containing 15 ml of sBHI and MIc, respectively. The resulting cultures were incubated at 35°C with shaking at 250 rpm, and aliquots were removed to determine the absorbance at 600 nm every 30 min for 6.5 h. Growth rates were calculated by nonlinear regression analysis.
To evaluate the growth of the dsbA mutant in comparison to the growth of the hbpA mutant under heme limitation conditions, strains RXV, RdsbAV, RhbpAV, and RhbpAC were grown in standing overnight cultures, washed once in sterile Hanks' balanced salt solution (HBSS) (Invitrogen, Carlsbad, CA), and diluted to obtain an OD600 of 0.01 in BHI broth supplemented with NAD and different concentrations (5, 0.5, 0.25, and 0.025 μg/ml) of heme (Sigma-Aldrich, St. Louis, MO) or hemoglobin (Becton, Dickinson and Company) in a 96-well microplate (final volume, 200 μl). The cultures were then incubated at 35°C in the microplate reader, and the absorbance at 600 nm was determined every 10 min for 16 h.
Growth of hbpA strains.
To compare the generation times obtained with different heme concentrations under anaerobic and aerobic conditions, overnight cultures of strains Rd, RdV, RhbpAV, and RhbpAC (pelleted and resuspended in HBSS) were used to inoculate 10 ml of BHI containing different concentrations of free heme (10, 0.5, 0.05, and 0 μg/ml). The cultures were then aliquoted into the wells of 11 96-well flat-bottom dishes. One dish was incubated at 35°C for 14 h in the microplate reader, and the absorbance at 600 nm was determined every 10 min (no aerobic growth was detected in wells not supplemented with heme). The other 10 dishes were sealed in individual BD GasPak EZ Anaerobe gas-generating pouches (Becton, Dickinson and Company) and incubated at 35°C. Dishes were removed from the pouches at appropriate intervals, and the absorbance at 600 nm was recorded. Growth rates were determined as described above.
Cultures were grown in triplicate as described above for the DTT sensitivity assay, and competent cells were prepared from these cultures as previously described (7
). The competence of mutant and parental strains was measured by assessing the transformation frequencies with chromosomal DNA from a streptomycin-resistant (Smr
) H. influenzae
strain (1 μg) and selection on sBHI agar plates containing 100 μg/ml streptomycin. Transformation efficiencies were calculated by dividing the number of Smr
colonies by the number of colonies on sBHI agar plates without antibiotic. Transformation frequencies were normalized by log10
transformation and analyzed with Prism 4.0c (GraphPad Software, San Diego, CA) using analysis of variance (ANOVA) with Bonferroni's multiple-comparison test to evaluate differences in frequency between RdsbAV and all other strains.
Murine bacteremia model.
Standing overnight cultures of strains having an OD600 of 0.01 were inoculated into 10 ml of sBHI in culture tubes. The resulting cultures were incubated in an anaerobic chamber with shaking at 120 rpm and 35°C for 5 h, conditions that were permissive for growth of the hbpA mutant. For coinfection, each experimental strain was mixed with the RdlacZ reference strain at a 1:1 ratio. Prior to inoculation, bacteria were washed and diluted in HBSS to obtain a final concentration of 2 × 109 bacteria per ml. Female 6.5-week-old C57BL/6J mice (four or five mice per strain; The Jackson Laboratory, Bar Harbor, ME) were inoculated by intraperitoneal (i.p.) injection of 200 μl of a bacterial suspension. Twenty-four hours after inoculation, 5 μl of blood was recovered aseptically from each mouse via tail bleeding. The blood was diluted into BHI broth, plated on sBHI agar plates for single-strain infections or on sBHI agar plates containing S-Gal (3,4-cyclohexenoesculetin β-d-galactopyranoside; Sigma-Aldrich) for coinfections, and incubated overnight in an anaerobic chamber at 35°C to determine the number of CFU. For statistical analysis, the numbers of CFU/ml for single-strain infections were normalized by log10 transformation for ANOVA using Prism 4.0c. The coinfection CFU data were log10 transformed, and the ratio of each experimental strain to RdlacZ was calculated and analyzed using Prism 4.0c. Comparisons of two data sets were performed using the t test, and comparisons of more than two data sets were performed using ANOVA with Bonferroni's multiple-comparison test. All procedures with animals were conducted in accordance with NIH guidelines and with prior approval by the University of Massachusetts Medical School Institutional Animal Care and Use Committee.
Infant rat infections.
H. influenzae type b-derived strains were inoculated using inocula from standing overnight cultures having an OD600 of 0.01 into 50 ml of sBHI in 50-ml Erlenmeyer flasks. Cultures were incubated with shaking at 120 rpm at 35°C to obtain an OD600 of 0.4. Cells were washed once and diluted in sterile HBSS to obtain a final concentration of 2 × 103 bacteria per ml. Five-day-old Sprague-Dawley rat pups (Charles River Laboratories, Boston, MA) were inoculated i.p. with 100 μl of strains HXV (n = 11) and HdsbAV (n = 11) or with HdsbAC (n = 12). Infants inoculated i.p. with each strain were returned to their mothers, and each group was housed separately. Blood (5 μl) was collected aseptically via tail bleeding at 12, 36, and 120 h postinoculation, diluted into BHI, and plated on sBHI agar plates for to determine the number of CFU as described above. For statistical analysis, ANOVA with Bonferroni's multiple-comparison test was used as described above.
HbpA Western blotting.
For analysis of HbpA, strains were inoculated using inocula from standing overnight cultures into duplicate 50-ml sBHI cultures in 50-ml flasks to obtain a starting density of 0.01 OD600 and were incubated at 35°C with shaking at 250 rpm. When cultures reached the log phase, 1 ml was removed and pelleted by centrifugation (18,000 × g for 5 min) for immunoblot analysis, and the remaining culture was used for RNA isolation as described below. After removal of the supernatant, the pellets were normalized by resuspension in an appropriate volume of HBSS. Cells (0.3 OD600 equivalents per lane) were then boiled in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer, and proteins were separated by 8% SDS-PAGE, followed by electrotransfer to Immobilon-P (Millipore, Billerica, MA). HbpA-HA was visualized by Western blotting using the primary antibody anti-HA1.1 (1:1000; Covance, Berkeley, CA) and the secondary antibody goat anti-mouse immunoglobulin G-horseradish peroxidase conjugate (1:5,000; Upstate, Lake Placid, NY). Equal sample concentrations were verified by Coomassie blue staining. HbpA-HA was quantified by generating a 10% dilution series of each protein sample and resolving proteins by 8% SDS-PAGE. HbpA-HA was then visualized by Western blotting as described above. HbpA levels were quantified by densitometry using ImageJ (National Institutes of Health, Bethesda, MD).
To quantify hbpA
mRNA, we isolated total RNA in parallel from the 50-ml cultures that were used for the HbpA Western blot analysis using the TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was then treated with DNase I (Ambion, Austin, TX), extracted with acid phenol, extracted with chloroform, and concentrated by ethanol precipitation. The RNA samples (total amount, 5 μg) were used as templates for cDNA synthesis with random primers (New England Biolabs, Beverley, MA) and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Quantitative reverse transcription PCR (qRT-PCR) was performed with iQ SYBR green Supermix (Bio-Rad Laboratories, Hercules, CA), and fluorescence was measured using the DNA Engine Opticon II system (MJ Research, Waltham, MA). One-tenth of each cDNA reaction mixture was used as a template for qRT-PCR performed with primers 5′hbpART (5′-ATGATTAATTTGTTATAATCCATAGA-3′) and 3′hbpART (5′-CAAGCTGCCAAAACAAGAGT-3′), which amplified the first 200 bp of hbpA
. Primers RpoA5′ (5′-GTAGAAATTGATGGCGTATTG-3′) and RpoA3′ (5′-TCACCATCATAGGTAATGTCC-3′) were used to amplify the RNA polymerase alpha subunit gene, rpoA
, as an internal reference. The real-time cycler conditions used have been described previously (72
Western blotting for assessment of binding of complement C3 and C4 activation products was performed as previously described (19
). Briefly, cultures of strains RXV, RdsbAV, and RdsbAC were grown as described above for HbpA Western blotting and then washed and suspended in HBSS containing 0.15 mM CaCl2
and 1 mM MgCl2
(final reaction mixture volume, 0.5 ml). Normal human serum (NHS) pooled from 12 healthy individuals was added to a final concentration of 2% and incubated for 30 min at 37°C, which was followed by differential treatment with 1 M methylamine (pH 11), which dissociates complement ester linkages but not amide-linked complement from target structures (19
). Bacteria were lysed in 1× SDS-PAGE sample buffer and analyzed by immunoblotting using primary antibodies to human C3 (Sigma-Aldrich, St. Louis, MO) and C4 (Biodesign, Saco, ME) and alkaline phosphatase-conjugated secondary anti-human antibodies as described previously (19
). No differences in the binding profiles of the strains to C3 or C4 subunits with and without methylamine treatment were observed.
Serum bactericidal assay.
The sensitivity of dsbA
mutants to serum was determined as previously described (57
). Briefly, triplicate cultures of strains RXV, RdsbAV, RdsbAC, and RdgalU were grown as described above for the DTT sensitivity assay. At log phase, 2,000 CFU from each culture was diluted in HBSS and incubated at 37°C for 30 min with or without 2% (final concentration) NHS in a 150-μl reaction mixture. To determine the number of CFU, 15 μl was plated on sBHI agar. Bacteria were also incubated in parallel with serum that had been previously inactivated by incubation at 56°C for 30 min.
Ten optical density units of cells grown as described above for anti-HA immunoblotting was harvested at log phase by centrifugation at 5,000 × g for 5 min. Before thiol modification of periplasmic proteins, the outer membrane was disrupted using the methods described in a PeriPreps periplasting kit (Epicenter, Madison, WI). Briefly, the cell pellets were resuspended in 2 ml of 200 mM Tris (pH 7.4), 1 mM EDTA, 20% sucrose, and 30 U of lysozyme (Sigma-Aldrich, St. Louis, MO) and incubated at room temperature for 5 min. After incubation, 3 ml of cold water was added, which was followed by 10 min of incubation on ice. Each 5-ml preparation was then divided in half; one half was treated with 5 mM EZ-Link maleimide-(ethylene oxide)2-biotin (MPB) (which added 525.23 Da per bond) (Pierce, Rockford, IL), and the other half was not treated. After incubation for 50 min at room temperature, the resulting spheroplasts and associated membranes were collected by centrifugation at 4,000 × g for 15 min and resuspended in 375 μl of Peripreps lysis buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, 1 mM EDTA, 0.1% deoxycholate). After lysis, equivalent 0.30 OD600 of each sample was boiled for 5 min in SDS loading buffer, and proteins were separated by nonreducing 8% SDS-PAGE. HbpA-HA was then visualized by Western blotting as described above. The apparent levels of HbpA-HA in the spheroplasts were similar to those in whole-cell lysates of the same number of cells (data not shown), suggesting that HbpA-HA is localized primarily in this fraction, which is consistent with membrane localization of the predicted HbpA lipoprotein.