INFECTION AND IMMUNITY, Apr. 2008, p. 1498–1508
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 4
The Periplasmic Disulfide Oxidoreductase DsbA Contributes to
Haemophilus influenzae Pathogenesis?
Charles V. Rosadini, Sandy M. S. Wong, and Brian J. Akerley*
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
Received 13 October 2007/Returned for modification 25 November 2007/Accepted 8 January 2008
Haemophilus influenzae is an obligate human pathogen that persistently colonizes the nasopharynx and
causes disease when it invades the bloodstream, lungs, or middle ear. Proteins that mediate critical interac-
tions with the host during invasive disease are likely to be secreted. Many secreted proteins require addition
of disulfide bonds by the DsbA disulfide oxidoreductase for activity or stability. In this study, we evaluated the
role in H. influenzae pathogenesis of DsbA, as well as HbpA, a substrate of DsbA. Mutants of H. influenzae Rd
and type b strain Eagan having nonpolar deletions of dsbA were attenuated for bacteremia in animal models,
and complemented strains exhibited virulence equivalent to that of the parental strains. Comparison of
predicted secreted proteins in H. influenzae to known DsbA substrates in other species revealed several proteins
that could contribute to the role of dsbA in virulence. One candidate, the heme transport protein, HbpA, was
examined because of the importance of exogenous heme for aerobic growth of H. influenzae. The presence of a
dsbA-dependent disulfide bond in HbpA was verified by an alkylation protection assay, and HbpA was less
abundant in a dsbA mutant. The hbpA mutant exhibited reduced bacteremia in the mouse model, and
complementation restored its in vivo phenotype to that of the parental strain. These results indicate that dsbA
is required in vivo and that HbpA and additional DsbA-dependent factors are likely to participate in H.
Haemophilus influenzae efficiently colonizes the human naso-
pharyngeal mucosa in a primarily asymptomatic manner, and the
carriage frequency is ?80% in healthy adults (48). However, it
can disseminate to other anatomical sites and cause otitis media,
ingitis in children (13, 22, 37, 44, 48–50, 63). The incidence of H.
influenzae meningitis has dramatically declined in populations
immunized with a vaccine against the type b capsular polysaccha-
ride. The vaccine has not affected the incidence of infection with
nontypeable H. influenzae (NTHi) strains, which lack the capsule.
otitis media but in rare cases can invade the bloodstream, leading
to meningitis. This disease profile raises the possibility that genes
promoting intravascular invasion could be present in NTHi
strains (15, 18, 53, 54). However, the molecular basis for the
invasive properties of H. influenzae that promote transmission
from the nasopharynx to the bloodstream or middle ear is not
Secreted bacterial proteins mediate critical aspects of patho-
genesis, including attachment, nutrient utilization, and subver-
sion of host defenses. Many secreted proteins of gram-negative
bacteria acquire disulfide bonds in the periplasm that stabilize
their mature, folded structures (9). Formation of such linkages
has been most extensively studied in Escherichia coli, in which
a series of disulfide oxidoreductases (Dsb) create and exchange
disulfide bonds in periplasmic proteins (for reviews, see refer-
ences 34 and 51). The soluble periplasmic disulfide oxi-
doreductase, DsbA, directly catalyzes this process by exchang-
ing its disulfide bond with free thiol groups of cysteine residues
in target proteins (21, 75). DsbA is efficiently reoxidized by
DsbB, a membrane protein that transfers electrons to quinones
for subsequent transfer to electron acceptors of the respiratory
chain (8, 51). The soluble periplasmic DsbC and DsbG pro-
teins mediate rearrangement of mispaired disulfides using
electrons transferred via the membrane-bound DsbD protein
from cytoplasmic thioredoxin (3, 10, 38, 58, 64, 76). Mutants
defective in periplasmic disulfide bond formation are viable
under standard culture conditions but exhibit a range of phe-
notypes as a result of defective maturation of secreted pro-
teins. The effects vary depending on the repertoire of periplas-
mic and secreted substrates of DsbA in different bacteria. The
deficiencies can involve single enzymes that require a disulfide
bond for activity, such as the periplasmic alkaline phosphatase,
PhoA, of E. coli, as well as defects in components of transport-
ers, resulting in inappropriate localization of substrates.
DsbA homologs contribute to the pathogenesis of multiple
bacterial species, in which they are required for maturation or
export of major secreted virulence factors. DsbA activity is re-
quired for production of functional type IV pili (also called fim-
briae) that mediate adherence to host surfaces in Vibrio cholerae,
Neisseria meningitidis, enteropathogenic E. coli, and uropatho-
genic E. coli (33, 55, 66, 77). Toxin production or secretion is
defective in many dsbA mutants; the toxins affected include chol-
era toxin in Vibrio cholerae, heat-labile and heat-stable E. coli
Type III secretion systems consist of multisubunit protein con-
duits that inject effector proteins directly from the bacterial cyto-
plasm into host cells to subvert diverse host cell functions. Com-
ponents of the type III secretion apparatus are defective in dsbA
mutants of Yersinia pestis, Shigella flexneri, Pseudomonas spp., and
Salmonella enterica serovar Typhimurium (26, 32, 42, 68). Fur-
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, University of Massachusetts Medical School,
55 Lake Ave., N., S6-242, Worcester, MA 01655. Phone: (508) 856-1442.
Fax: (508) 856-1422. E-mail: Brian.Akerley@umassmed.edu.
?Published ahead of print on 22 January 2008.
thermore, DsbA has been implicated in systemic infection by E.
coli K1 and prolonged survival of adherent-invasive E. coli within
macrophages (12, 23).
The dsbA gene of H. influenzae (HI0846, also called por)
transcomplements a dsbA mutant of E. coli (67). Disruption of
dsbA with a transposon insertion resulted in changes in se-
creted protein localization in a cellular fractionation experi-
ment and dramatically reduced the natural transformation ef-
ficiency (67). The role of DsbA in H. influenzae pathogenesis
has not been examined. However, a transposon-based “signa-
ture-tagged mutagenesis” screen detected the putative dsbB
homolog as a virulence gene candidate in an infant rat model
of bacteremia, suggesting a potential role for periplasmic di-
sulfide bond formation in H. influenzae pathogenesis (28). In
H. influenzae, the protein targets of DsbA and virulence factors
dependent on its activity have not been identified. In this study,
we demonstrated that dsbA is required for H. influenzae bac-
teremia caused by both unencapsulated strain Rd and a viru-
lent encapsulated type b strain. Heme uptake is required for
aerobic growth of H. influenzae, which cannot synthesize the
porphyrin ring (24, 70), and several heme utilization pathways
have been implicated in bloodstream infection by H. influenzae
(47, 61). We demonstrate that the heme transport protein,
HbpA, contains a DsbA-dependent disulfide bond. A nonpolar
hbpA deletion mutant caused reduced bacteremia in mice, yet
the defect was not as pronounced as that of the dsbA mutant.
Based on these results, it is likely that dsbA is required in vivo
for production of optimal levels of hbpA and that additional
virulence factors that remain to be identified also participate in
the critical role of DsbA in H. influenzae pathogenesis.
MATERIALS AND METHODS
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) supple-
mented with 10 ?g/ml hemin and 10 ?g/ml NAD (sBHI), in MIc, a low-nutrient
medium capable of supporting growth of H. influenzae (7), or on sBHI agar plates at
35°C. Development of competence for transformation of H. influenzae was accom-
plished as previously described (7). For selection of Rd- and Hib-derived strains,
antibiotics were used at the following concentrations: 8 ?g/ml tetracycline, 20 ?g/ml
kanamycin, 10 ?g/ml gentamicin, and 100 ?g/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 (71). 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 and xylB were confirmed by perform-
ing PCR with primers specific for sequences outside the inserted recombinant
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
with primers F-NTdsb (5?-AAAGATCTGCTCTTCAATGAAAAAAGTATTACT
TGC-3?) and 3dsbAHA (5?-AAAGATCTGCTCTTCGTAATGCATAATCTGGC
ACATCATATGGATATTTTTGCAATAAACCTTTTACGGTT-3?), which intro-
duced 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?-TT
CTTTCCTCTTATTTAATGATACCGCGAG-3?]), the 569-bp aacC1 gene encod-
ing gentamicin resistance (primers 5GentD [5?-TAAATAAGAGGAAAGAAATG
TTACGCAGCAGCAACGATGTT-3?] and 3GentD [5?-CATTAAACCAATTTT
TCGTTAGGTGGCGGTACTTGGGTCGAT-3?]), and the 1,641-bp 3? region
starting at the dsbA termination codon (primers 5Dsbout [5?-CGAAAAATTGGT
TTAATGCCAGCCC-3?] and 3848H [5?-TTTAAGCTTCTACTTGCGAATGAG
CCATAGGC-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-induc-
ible 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
TAAACCTTTTACGGT-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?-GAGGCCCGTGTCTCAAAATC
TCTGATG-3?) and 3revRfaD1 (5?-AACAGGCTACGATAAACCATTCAAA
ACAGT-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
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?-ACGTTTATCAACAGCGATAGGATC
AAGT-3?) and 3pDsbAsapKan (5?-CATCAGAGATTTTGAGACACGGGCCTC
GATACCGCGA-3?) were used instead of primers pXT10thyA-F and 3dsbAkan.
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 Kmrtransfor-
mants 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 se-
quence 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?-AGTCATTCACGCCAGTTGGCACTGGA
T-3?) and 3hbp1 (5?-TTCCCGTTGAATATGGCTCATACCTCAATGTTAGG
CAGGGAATGCCCTA-3?), an 816-bp PCR product containing the coding re-
gion for the kanamycin resistance gene generated with primers 5kan1.1 (5?-AT
GAGCCATATTCAACGGGAA-3?) and 3kan1.1 (5?-TTAGAAAAACTCATC
GAGCATCAAATG-3?), and a 1,020-bp PCR product containing the 3? flanking
region of hbpA generated with primers 5hbp3 (5?-CATTTGATGCTCGATGA
GTTTTTCTAATTCATATTGATTTACTTATTTTAAGCCCT-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 Kmrtransformants 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
VOL. 76, 2008 DsbA AND H. INFLUENZAE PATHOGENESIS 1499
with linearized vector pXT10, and Tetrtransformants were isolated to create
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?-AAAGCT
CTTCAATGATTAATTTGTTATAATCCATAGA-3?) and 3hbpha (5?-TTTGCT
CTCACACCATA-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 Tetrto 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 Gmrtransformants were
selected to create strain RhbpAC?dsbA.
Other strains. 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 Kmrcassette. For all mutant strains, replace-
ment 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 OD600of 0.02, and 100 ?l was transferred
to a 96-well flat-bottom dish. Each well in the dish was then treated with 100 ?l
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 absor-
bance 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 dele-
tion 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 OD600of
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 OD600of 0.02, and
100 ?l of each culture was seeded into a 96-well flat-bottom dish. Hydrogen
peroxide (Sigma-Aldrich, St. Louis, MO) diluted in 100 ?l of sBHI was then
added to cultures grown in 25-ml flasks at final concentrations of 0, 62.5, 125, and
250 ?M in sBHI and to anaerobically grown cultures at final concentrations of 0,
62.5, 125, and 500 ?M. 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 OD600of 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
to obtain an OD600of 0.01 in BHI broth supplemented with NAD and different
concentrations (5, 0.5, 0.25, and 0.025 ?g/ml) of heme (Sigma-Aldrich, St. Louis,
MO) or hemoglobin (Becton, Dickinson and Company) in a 96-well microplate
(final volume, 200 ?l). 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 concentra-
tions of free heme (10, 0.5, 0.05, and 0 ?g/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
Competence assay. 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 mea-
sured by assessing the transformation frequencies with chromosomal DNA from a
streptomycin-resistant (Smr) H. influenzae strain (1 ?g) and selection on sBHI agar
plates containing 100 ?g/ml streptomycin. Transformation efficiencies were calcu-
lated by dividing the number of Smrcolonies 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
OD600of 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 ? 109bacteria 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 ?l of a
bacterial suspension. Twenty-four hours after inoculation, 5 ?l 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 ?-D-galactopyrano-
side; 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 log10trans-
formation 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 OD600of 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 OD600of 0.4. Cells were washed once and diluted in
sterile HBSS to obtain a final concentration of 2 ? 103bacteria per ml. Five-
day-old Sprague-Dawley rat pups (Charles River Laboratories, Boston, MA)
were inoculated i.p. with 100 ?l 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 ?l) 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 OD600and 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 OD600equivalents per lane) were then
boiled in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
(PAGE) sample buffer, and proteins were separated by 8% SDS-PAGE, fol-
lowed 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).
qRT-PCR. 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
1500 ROSADINI ET AL.INFECT. IMMUN.
I (Ambion, Austin, TX), extracted with acid phenol, extracted with chloroform,
and concentrated by ethanol precipitation. The RNA samples (total amount, 5
?g) were used as templates for cDNA synthesis with random primers (New
England Biolabs, Beverley, MA) and SuperScript II reverse transcriptase (In-
vitrogen, 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?-ATG
ATTAATTTGTTATAATCCATAGA-3?) and 3?hbpART (5?-CAAGCTGCCA
AAACAAGAGT-3?), which amplified the first 200 bp of hbpA. Primers RpoA5?
(5?-GTAGAAATTGATGGCGTATTG-3?) and RpoA3? (5?-TCACCATCATA
GGTAATGTCC-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).
Complement binding. Western blotting for assessment of binding of comple-
ment C3 and C4 activation products was performed as previously described (19,
56). 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 CaCl2and 1 mM MgCl2(final reaction mixture
volume, 0.5 ml). Normal human serum (NHS) pooled from 12 healthy individ-
uals 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, 56). 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 previ-
ously (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 de-
termined 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-?l reaction mixture. To determine the number of CFU, 15 ?l 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.
Thiol modification. 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 sphero-
plasts and associated membranes were collected by centrifugation at 4,000 ? g
for 15 min and resuspended in 375 ?l 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 OD600of 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.
Phenotypic properties of a nonpolar dsbA deletion mutant.
A series of strains were constructed to evaluate the potential
role of DsbA in H. influenzae pathogenesis (Table 1). We first
TABLE 1. Strains and plasmids used in this work
Plasmid or strain Relevant features, genotype, and/or descriptionReference
pXT10 Delivery vector for chromosomal expression at the xylose locus of H. influenzae containing xylF,
xylB, xylA?4-804, and the tetAR tetracycline resistance cassette
pXT10 carrying dsbA expressed from the xylA promoter
pXT10 carrying dsbA expressed from the dsbA promoter
pXT10 carrying an HA-tagged hbpA gene expressed from the hbpA promoter
Wild type; H. influenzae capsule-deficient type d
Rd xylA?4-804::tetAR; Rd carrying empty Tetrvector sequence from pXT10 inserted at the xyl
locus in place of xylA
Rd xylA?4-804::dsbA; Rd carrying dsbA expressed via the xylA promoter from pXyldsbA1.1
RX dsbA::aacC1; dsbA deletion mutant carrying dsbA expressed via the xylA promoter from
pXyldsbA1.1 replacing xylA
Rd xylA?4-804::aphI; Rd carrying the Kmrcassette replacing xylA
RdsbAX xylA?4-804::aphI; dsbA deletion mutant carrying the Kmrcassette replacing xylA
RdsbAX xylA?4-804::dsbA; dsbA mutant complemented with dsbA expressed via the dsbA
promoter from pDsbA1.2 replacing xylA
hbpA::aphI; hbpA deletion mutant
RhbpA xylA?4-804::tetAR; hbpA deletion mutant carrying empty Tetrvector sequence from
pXT10 replacing xylA
RhbpAV xylA?4-804::hbpA; hbpA mutant carrying hbpA expressed via the hbpA promoter from
pXhbp1.5 replacing xylA
RhbpAC dsbA::aacC1; hbpA dsbA double mutant carrying hbpA expressed via the hbpA
promoter from pXhbpA1.5 replacing xylA
Rd xylA?4-804::lacZ; Rd carrying a copy of lacZ replacing xylA
Wild type; H. influenzae type b Eagan
Hib xylA?4-804::aphI; Hib carrying the Kmrcassette replacing xylA
Hib dsbA::aacC1 xylA?4-804::aphI; dsbA deletion mutant carrying the Kmrcassette replacing xylA
Hib dsbA::aacC1 xylA?4-804::dsbA; dsbA mutant complemented with dsbA expressed via the
dsbA promoter from pDsbA1.2 replacing xylA
RdsbAX This study
VOL. 76, 2008 DsbA AND H. INFLUENZAE PATHOGENESIS 1501
verified that the dsbA mutant exhibited the DTT sensitivity
phenotype previously seen with dsbA mutants of other species
(43), and we determined its growth properties. Wild-type par-
ent strain Rd, Rd carrying the “empty vector” (RXV), a dsbA
deletion mutant carrying the “empty vector” (RdsbAV), and
the complemented strain (RdsbAC) were evaluated to deter-
mine their growth under a range of conditions and to deter-
mine defects in DTT resistance and transformation. The gen-
eration times under aerobic conditions in rich medium (sBHI)
for RXV, RdsbAV, and RdsbAC were 32 ? 2, 32 ? 3, and
36 ? 4 min, respectively, and the generation times in defined
medium (MIc) were 48 ? 4, 41 ? 2, and 38 ? 3 min, respec-
tively. Similarly, the growth yields of these strains after 6.5 h of
anaerobic growth in sBHI were indistinguishable. The growth
yields of all DsbA?strains (Rd, RXV, and RdsbAC) were
equivalent after 16 h in the presence of 5 mM DTT, with final
average densities of ?0.5 OD600, whereas the growth of the
DsbA?strain, RdsbAV, was dramatically attenuated under
these conditions and the density did not exceed 0.1 OD600,
similar to results obtained with dsbA mutants of E. coli (43).
Strain RdsbAX, which contains a D-xylose-inducible copy of
dsbA and was used to construct strains RdsbAV and RdsbAC,
was resistant to DTT in the presence of 1 mM D-xylose and
sensitive to DTT in the absence of D-xylose.
H. influenzae dsbA (por) was previously implicated in natural
transformation. Therefore, we evaluated the transformation
efficiencies of our strains using H. influenzae DNA carrying a
streptomycin resistance allele. The transformation efficiencies
relative to the wild-type parental strain Rd for strains RXV,
RdsbAV, and RdsbAC, were 1.12, 1.33 ? 10?6, and 1.04,
respectively, and the 6-log-lower transformation frequency of
the dsbA mutant (RdsbAV) relative to the other strains was
statistically significant (P ? 0.0001). Therefore, previously re-
ported phenotypes associated with dsbA mutants were ob-
served with our in-frame H. influenzae dsbA deletion mutants,
and complemented strains exhibited the wild-type phenotypes.
DsbA is required during H. influenzae infection in mice. The
strains generated as described above provided a well-defined
set of mutants for investigation of the role of dsbA during
infection. Although not a recent clinical isolate, Rd has viru-
lence properties similar to those of clinically important NTHi
strains in several models of infection and has provided a useful
system for studies of H. influenzae biology and pathogenesis
(16, 40). The mouse model was used to evaluate bloodstream
survival of the dsbA mutant, RdsbAV, compared to that of the
vector-only control strain, RXV. At 24 h postinoculation, 48-
fold-fewer bacterial CFU were recovered from mice inoculated
with the dsbA mutant than from mice inoculated with the
control strain (Fig. 1A). The level of bacteria recovered from
most of the mice inoculated with RdsbAV was close to the
limit of detection. An additional experiment was conducted to
confirm this result with a complemented strain, RdsbAC. To
further assess the level of attenuation, this experiment was
performed as a competition between each strain and strain
RdlacZ, which expresses E. coli lacZ at the xyl locus, using
mixed infections. Consistent with results obtained from single-
strain inoculations, the competitive index of RdsbAV was 100-
to 170-fold less than that of Rd, RXV, or RdsbAC (Fig. 1B).
Therefore, infection with a mutant containing a nonpolar dsbA
deletion resulted in reduced levels of bacteremia in mice, and
complementation verified that this effect was specific to the
Pathogenesis-associated phenotypes of the dsbA mutant. H.
influenzae lacks previously implicated dsbA-dependent viru-
lence factors found in other species, including exotoxins and
type III secretion structures. Production of the type IV pilus
found in some NTHi strains is likely to require dsbA; however,
the pilus gene cluster is absent in Rd (20). Therefore, we
examined several major virulence-associated phenotypes of H.
influenzae to determine whether a defect in a known patho-
genic mechanism could account for the survival defect of the
dsbA mutant in vivo.
Resistance to oxidative stress generated by hydrogen perox-
ide exposure has been correlated with H. influenzae pathogen-
esis in several studies (17, 72). Therefore, we addressed the
possibility that loss of DsbA confers sensitivity to this oxidant.
After exposure to either anaerobic or aerobic pregrowth con-
ditions, the mutant and the wild type exhibited equal levels of
growth inhibition during exposure to hydrogen peroxide at a
range of doses (data not shown).
Multiple structures of the lipooligosaccharide (LOS) outer
core have been implicated in animal models of H. influenzae
bacteremia (25, 31, 60), and resistance to complement has
emerged as an important virulence mechanism mediated by
these structures (19, 30). Therefore, we investigated whether
the dsbA mutant exhibits major LOS structural alterations or
increased susceptibility to killing by serum complement. We
detected no apparent differences in LOS mobility on SDS-
PAGE gels for the dsbA mutant and the wild type. Wild-type,
dsbA mutant, and complemented strains were compared using
a serum bactericidal assay with 2% pooled NHS. The levels of
survival for strains RdV, RdsbAV, and RdsbAC were 12, 2.9,
and 11.6%, respectively (P ? 0.05). For comparison, a galU
mutant deficient in synthesis of the LOS outer core, a structure
predicted to be essential for complement resistance, was tested
in parallel and exhibited 0% survival. No killing of H. influen-
zae was observed with heat-inactivated serum, consistent with
an essential role for complement in this assay. Differences in
levels of complement binding to strains RdV, RdsbAV, and
FIG. 1. Effect of dsbA mutation on survival of H. influenzae in the
mouse model of bacteremia. Strains were inoculated i.p. into mice, and
bacteremia was assessed after 24 h. The symbols indicate data for
individual animals, and the dashed lines indicate the averages. The
lower limit of detection (LLD) was 500 CFU/ml. Values less than the
lower limit of detection indicate that bacteremia was not detected.
(A) Single-strain infection. The asterisk indicates that the P value is
0.039 (t test). (B) Coinfections with the experimental strains indicated
and reference strain RdlacZ. The competitive index is the ratio of the
LacZ?experimental strain to LacZ?reference strain RdlacZ. The
asterisk indicates that the P value is ?0.001 (ANOVA with Bonferroni’s
1502ROSADINI ET AL.INFECT. IMMUN.
RdsbAC were not detected, as assessed on anti-C3 and anti-C4
immunoblots containing lysates of cells that had been incu-
bated with 2% pooled NHS (data not shown), although it is
possible that a small difference in C3 or C4 binding not de-
tected by immunoblotting could have mediated the moderate
increase in serum sensitivity observed for the mutant.
We concluded that the hydrogen peroxide resistance and
LOS production of the dsbA mutant were not markedly im-
paired under the conditions tested. An effect on serum resis-
tance that could play a role was observed. However, this effect
was moderate, and it seems likely that DsbA influences addi-
tional factors required for virulence. To address this hypothe-
sis, we sought the identities of potential DsbA substrates in H.
influenzae. Proteins containing DsbA-dependent disulfide
bonds have been identified in E. coli (29, 35, 39). These pro-
teins were compared by BLASTP (1) (http://www.ncbi.nlm.nih
.gov) to the predicted proteins in the H. influenzae genome to
derive a list of potential DsbA targets in H. influenzae (Table
2). The H. influenzae proteins identified by this search include
a predicted periplasmic lipoprotein, HbpA, which is required
for utilization of multiple heme sources (27, 45). Multiple
systems participate in scavenging heme from sources in the
host that include heme-hemopexin, hemoglobin, hemoglobin-
haptoglobin, heme-albumin, and free heme (14, 46). HbpA
appears to be required for scavenging low levels of heme re-
gardless of the source or carrier protein, suggesting that it
could be critical for growth in vivo. The link between DsbA and
HbpA suggested a potential mechanism of attenuation of the
dsbA mutant in the mouse model.
Heme uptake protein HbpA is a target of disulfide oxi-
doreductase. Based on comparison to the crystal structure of
the highly related E. coli DppA protein, which has an intramo-
lecular disulfide bond (52), HbpA has a predicted disulfide
bond between cysteine residues Cys27 and Cys255 of the ma-
ture protein. A third cysteine located at the N terminus con-
stitutes the predicted lipoprotein acylation site. Many proteins
containing DsbA-dependent disulfide bonds are less stable in
DsbA-deficient cells. Therefore, we examined the effect of the
dsbA deletion mutation on levels of HbpA. To address this
question, we developed a functional derivative of HbpA fused
to an epitope tag from the influenza virus HA (HbpA-HA).
We first constructed a nonpolar hbpA deletion mutant
(RhbpAV) of H. influenzae. The mutant was defective for
aerobic growth on medium containing low levels of heme, as
previously reported for an independently derived hbpA inser-
tional mutant (45). Furthermore, the hbpA mutant exhibited
anaerobic growth equivalent to that of wild-type strain Rd and
the isogenic “vector-only” strain (RdV), regardless of heme
availability (Table 3). When expressed in the hbpA mutant
background, HbpA-HA fully complemented the mutant for
aerobic growth at all heme concentrations tested (strain
RhbpAC), suggesting that the epitope tag does not impair its
function (Table 3).
The resulting strains were used to assess the effect of a dsbA
mutation on levels of HbpA-HA on Western blots, and tran-
script levels were assessed in parallel by qRT-PCR. The levels
of HbpA-HA in the dsbA deletion mutant RhbpAC?dsbA
were approximately 50% of the levels detected in the DsbA?
TABLE 2. Potential DsbA targets
Putative DsbA target
in E. coli
Known or proposed function in E. coli Potential H. influenzae homolog BLAST identity (%)Expect value
ArtJArginine ABC transporter periplasmic
Dipeptide/heme binding protein
Organic solvent tolerance protein
Outer membrane porin
Oligopeptide transporter periplasmic
Zinc uptake system periplasmic
ArtI (HI1179) 115/244 (47)7e-57
ZnuA ZnuA (HI0119) 140/341 (41)6e-70
TABLE 3. Growth phenotypes of hbpA mutants
Generation time (min)
Aerobic conditions with free heme supplement at a
Anaerobic conditions with free heme supplement at a concn ofb:
10 ?g/ml0.5 ?g/ml0.05 ?g/ml 10 ?g/ml 0.5 ?g/ml0.05 ?g/ml0 ?g/ml
46 ? 3
45 ? 1
41 ? 3
46 ? 3
47 ? 3
45 ? 2
55 ? 7
48 ? 2
57 ? 4
50 ? 3
53 ? 1
57 ? 8
65 ? 13
49 ? 4
61 ? 4
56 ? 1
62 ? 7
54 ? 2
60 ? 2
61 ? 2
65 ? 6
55 ? 4
66 ? 1
64 ? 3
73 ? 3
56 ? 5
66 ? 2
aGrowth was determined with a microplate reader.
bGrowth was determined in an anaerobic chamber.
cNG, no growth.
VOL. 76, 2008 DsbA AND H. INFLUENZAE PATHOGENESIS 1503
control strain, RhbpAC, as determined by densitometry (Fig.
2A). qRT-PCR detected no differences in the levels of hbpA-
specific transcripts in these cultures (data not shown). To-
gether, these results suggest that the effect of DsbA on HbpA
abundance is mediated at a posttranscriptional level, consistent
with its role as a disulfide oxidoreductase.
To more directly assess the role of DsbA in formation of di-
sulfide bonds in HbpA, the HbpA-HA protein was analyzed by
nonreducing SDS-PAGE after isolation from the DsbA?and
DsbA?H. influenzae strains (Fig. 2B). Whereas HbpA-HA from
DsbA?cells appeared as a single band, samples from DsbA?
cells yielded an additional HbpA-HA band with lower electro-
phoretic mobility. Treatment of cells prior to protein isolation
with a thiol reactive ligand, MPB, resulted in no change in
HbpA-HA in the parental strain (Fig. 2B, lanes 1 and 2), as
expected if the two nonacylated cysteine residues were in the
oxidized state as a disulfide bond. In contrast, the more slowly
migrating species in the dsbA mutant (Fig. 2B, lane 3) exhibited
an additional decrease in mobility in samples from MPB-treated
cells (Fig. 2B, lane 4), consistent with addition of MPB to free
thiols on cysteine residues of this protein. Relatively low levels of
the reduced form of HbpA were detected, consistent with de-
creased stability of the reduced form relative to the oxidized form
in the dsbA mutant, a characteristic property of many DsbA-
dependent proteins (9). Therefore, a longer exposure time was
used to clearly visualize the reduced form in Fig. 2B, masking the
decrease in total HbpA levels that was detected in the dsbA
mutant in the quantitative studies described above (Fig. 2A).
HbpA in the oxidized form was detected in the dsbA mutant,
and it is likely that some HbpA activity was retained in this
mutant. Consistent with this observation, we could not detect a
growth defect of the dsbA mutant on low-heme media. The
growth rates of DsbA?and DsbA?strains (RXV and RdsbAV)
were compared to those of the hbpA mutant and complemented
strains (RhbpAV and RhbpAC) with 5, 0.5, 0.25, and 0.025 ?g/ml
of either heme or heme-hemoglobin (data not shown). RhbpAV
exhibited progressively lower growth rates as the heme or hemo-
globin concentration was decreased, and RhbpAC grew at the
same rates as the wild type, similar to results shown in Table 3.
Conversely, no differences between RXV and RdsbAV were ob-
served, suggesting that the residual levels of active HbpA in the
dsbA mutant were sufficient for acquisition of these heme sources
in vitro. Together, these data indicate that the DsbA disulfide
oxidoreductase is required to maintain the complete oxidation of
free thiols on HbpA and for wild-type levels of this protein in H.
HbpA is required during bloodstream infection. Heme is
required for aerobic growth and is obtained by H. influenzae
from sources within the host. The decreased levels of HbpA
observed in the dsbA mutant could have contributed to de-
creased survival of this strain in the bloodstream by interfering
with heme acquisition in vivo, where heme is efficiently seques-
tered by multiple systems of the host. To evaluate this hypoth-
esis, we assessed the role of hbpA in the mouse model using the
hbpA mutant RhbpAV, the isogenic HpbA?parent strain
RdV, and the complemented strain RhbpAC. Inocula were
prepared from cultures grown anaerobically, conditions which
were permissive for growth of the hbpA mutant (Table 3), and
mice were inoculated by the i.p. route (Fig. 3). In single-strain
infections there was a decrease in the number of bacterial CFU
recovered from mice inoculated with RhbpAV compared to
mice inoculated with RdV (17-fold) or RhbpAC, (?60-fold);
however, the trend was not statistically significant (Fig. 3A). To
control for variation between animals, we repeated the exper-
iment using the competition format. Each strain was coinocu-
lated with an equal number of cells of strain RdlacZ, and
competitive indices were evaluated. The mutant exhibited a
?27-fold defect in competition relative to the “vector-only”
FIG. 2. Effects of dsbA mutation on HbpA protein levels and thiol
redox state. (A) Detection of HbpA levels in DsbA?and DsbA?
strains. Whole-cell lysates of duplicate cultures of RhbpAC (comple-
mented hbpA deletion mutant carrying hbpA-HA in the xyl locus)
(lanes 1 and 2) and RhbpAC?dsbA (RhbpAC with dsbA deletion)
(lanes 3 and 4) were resolved by 8% SDS-PAGE under reducing
conditions and detected by anti-HA Western blotting (left panel).
Equal sample concentrations were verified by Coomassie blue staining
(right panel). (B) Differential modification of thiols on HbpA in the
dsbA mutant compared to the wild type. Spheroplasts were prepared
from log-phase cultures, resolved by 8% SDS-PAGE under nonreduc-
ing conditions, and detected by anti-HA Western blotting. The strains
were the same as those used for panel A and were treated (?) or not
treated (?) with 5 mM MPB as indicated. The arrows indicate the
oxidized form of HbpA (ox), the reduced form of HbpA (red), and the
reduced form of HbpA with thiols modified with MPB (red ? MPB).
FIG. 3. Effect of hbpA mutation on survival of H. influenzae in the
mouse model of bacteremia. Strains were inoculated i.p. into mice, and
bacteremia was assessed after 24 h. The symbols indicate data for
individual animals, and the dashed lines indicate the averages. The
lower limit of detection (LLD) was 500 CFU/ml. Values less than the
lower limit of detection indicate that bacteremia was not detected.
(A) Single-strain infection. (B) Coinfections with the experimental
strains indicated and reference strain RdlacZ. The competitive index is
the ratio of the LacZ?experimental strain to LacZ?reference strain
RdlacZ. The asterisk indicates that the P value is ?0.001.
1504 ROSADINI ET AL.INFECT. IMMUN.
and complemented strains, and the differences were statisti-
cally significant (Fig. 3B). We concluded that survival of the
hbpA mutant is attenuated in the bacteremia model, but to a
lesser extent than survival of the dsbA mutant. Therefore, a
decreased level of HbpA could contribute to the defect in dsbA
mutants during infection, yet additional factors, such as serum
sensitivity and other mechanisms that remain to be identified,
are likely involved.
DsbA is required for growth and persistence of virulent H.
influenzae type b in the bloodstream. We next addressed
whether dsbA is required during infection by the highly virulent
organism H. influenzae type b strain Eagan. The infant rat
bacteremia model provides a well-characterized system for ex-
amining factors required for H. influenzae type b pathogenesis.
Therefore, the mutations used to evaluate the role of dsbA in
Rd were moved into the Hib background. Infant rats that were
5 days old were inoculated i.p. with wild-type “vector-only”
strain HXV, dsbA mutant HdsbAV, and complemented strain
HdsbAC, and bloodstream infection was monitored at 12, 36,
and 120 h postinoculation (Fig. 4). At all sampling times, the
number of H. influenzae CFU recovered from animals inocu-
lated with the dsbA mutant was at least 100-fold lower than the
number of H. influenzae CFU recovered from animals inocu-
lated with the parental or complemented strain, and the level
of attenuation was statistically significant in all cases. Further-
more, by 120 h postinoculation only 2 of 11 animals inoculated
with the dsbA mutant had detectable bacteremia, whereas most
of the animals infected with the wild-type strain (9/11) or the
complemented strain (11/11) remained infected, with mean
bacterial levels of 9.5 ? 104and 5.6 ? 104CFU/ml, respec-
tively. These results indicate that dsbA is required for efficient
production and persistence of a high level of bacteremia in the
infant rat model with a virulent clinical isolate of H. influenzae
We report a role for the H. influenzae disulfide oxidoreduc-
tase, DsbA, in bloodstream infection. Nonpolar dsbA deletion
mutations in either the strain Rd or H. influenzae type b strain
Eagan background resulted in equal levels of attenuation in
animal models, and the virulence of complemented strains was
equivalent to that of the parental strains. Because the in vivo
defect was observed with dsbA mutants of both nonencapsu-
lated strain Rd and an encapsulated H. influenzae type b strain,
an effect on production of capsule would be unlikely to account
for these observations. Therefore, we investigated several
other potential mechanisms. The primary set of factors impli-
cated in pathogenesis of nonencapsulated H. influenzae in an-
imal models includes genes involved in LOS synthesis, evasion
of complement deposition, and oxidative stress resistance. We
detected no apparent role for dsbA in LOS synthesis or hydro-
gen peroxide resistance, although we cannot exclude the pos-
sibility that these phenotypes are influenced in a subtle way
that our assays were not sufficiently sensitive to detect. A
decrease in serum resistance was observed in the dsbA mutant,
and it will be of interest to establish the mechanism by which
DsbA contributes to this virulence-related trait. However, the
effect on serum resistance was only moderate and does not
seem to be sufficient to account for the full defect of the dsbA
mutant in pathogenesis. The results suggest that an unrecog-
nized factor(s) may account for the observed virulence defect
of the dsbA mutants.
To expand our search to other factors that could participate
in the defect of the dsbA mutant in vivo, we considered a set of
potential secreted substrates of H. influenzae DsbA identified
by comparison of amino acid sequences to reported DsbA
targets in other species. The resulting list of potential DsbA
substrates in H. influenzae includes a number of known or
suspected nutrient transport proteins. Therefore, a nutritional
deficiency could contribute to the defect of the dsbA mutant in
the blood. We examined growth of the H. influenzae dsbA
mutant under a range of in vitro conditions. The H. influenzae
dsbA mutant grew normally under aerobic and anaerobic con-
ditions and in a low-nutrient medium. The only in vitro con-
dition under which we could detect a growth defect for the
dsbA mutant involved the presence of a high concentration (5
mM) of DTT, a condition that H. influenzae is unlikely to
encounter in vivo. Reducing agents are present in plasma and
include glutathione, which occurs as a mixture of reduced and
oxidized forms with a total concentration estimated to be ?5 to
30 ?M (4). Growth in the presence of glutathione was tested
FIG. 4. Effect of dsbA mutation on the virulence of H. influenzae type b in infant rats. Strains were inoculated i.p. into 5-day-old infant rats.
The symbols indicate data for individual animals, and the dashed lines indicate the averages. An asterisks indicates that the P value is ?0.001. The
lower limit of detection (LLD) was 500 CFU/ml. Values less than the lower limit of detection indicate that bacteremia was not detected.
VOL. 76, 2008DsbA AND H. INFLUENZAE PATHOGENESIS1505
using concentrations ranging from 0.005 to 1 mM, and growth
of the dsbA mutant and growth of the parental strain were
equivalent under each of these conditions (data not shown). A
general growth defect or sensitivity to physiological levels of
reducing agents does not appear to account for the decreased
virulence of our dsbA deletion mutants. If the effect of dsbA on
pathogenesis involves a defect in nutrient uptake or utilization,
then it is likely to involve a nutrient that is selectively limiting
in H. influenzae’s environment within the host.
One essential factor that H. influenzae cannot synthesize and
must obtain from the host is the porphyrin ring of heme. The
results of an amino acid sequence comparison of H. influenzae
proteins with known or probable DsbA substrates in other
species identified the H. influenzae heme binding protein,
HbpA, as a potential substrate of DsbA. Deletion of hbpA
results in an aerobic growth defect on media containing low
levels of exogenous heme sources (27, 45) and normal growth
under anaerobic conditions (Table 3). We identified the pres-
ence of a DsbA-dependent disulfide bond in HbpA and de-
creased abundance of HbpA in the dsbA mutant. Therefore,
we evaluated the phenotype of an hbpA mutant during infec-
tion. The hbpA mutant exhibited a defect in the murine bac-
teremia model, although it was not as pronounced as the defect
of the dsbA mutant. Complementation restored the ability of
the hbpA mutant to cause bacteremia at a level similar to that
observed with the parental strain. These results suggest that
decreased levels of HbpA could contribute to the in vivo defect
of the dsbA mutants.
The decreased level of HbpA in the dsbA mutant would be
expected to influence growth under heme limitation condi-
tions; however, we were unable to detect such an effect in vitro.
It is likely that residual HbpA activity in the dsbA mutant is
capable of supporting in vitro growth with low levels of heme.
Nevertheless, both DsbA and HbpA participate in blood-
stream infection. For survival in vivo, where diverse host fac-
tors efficiently sequester free heme, there may be a more strin-
gent requirement for wild-type levels of HbpA than there were
the in vitro conditions tested here. Alternatively, it is possible
that the in vivo growth defect of the dsbA mutant resulted from
effects on a DsbA-dependent protein whose role in virulence
remains to be identified. The partial attenuation of the hbpA
mutant compared to the more dramatic virulence defect of the
dsbA mutant supports the hypothesis that other factors are
involved. In this regard, two potential DsbA targets in H.
influenzae, Pzp1 (ZnuA) and outer membrane protein P5 (Ta-
ble 2), are required for growth under zinc-limiting conditions
in vitro (41) and adhesion to mucosal epithelium during colo-
nization of the chinchilla nasopharynx (11), respectively. In
addition, homologs of Pzp1 and P5 in several other bacterial
species have been implicated in pathogenesis (2, 36, 59, 62, 69,
73). We did not observe a requirement for zinc supplementa-
tion for in vitro growth of the dsbA mutant (data not shown),
potentially due to residual activity of Pzp1, yet is possible that
the zinc levels available to bacteria within the mammalian host
are lower than those in vitro. A defect in the level or activity of
Pzp1 in the H. influenzae dsbA mutant could contribute to its
virulence defect, and additional studies are required to evalu-
ate this hypothesis. In addition, a role for P5 during H. influ-
enzae bacteremia has not been reported, and it will be inter-
esting to investigate this possibility. Related outer membrane
proteins in other pathogens have been implicated in diverse
aspects of pathogenesis, including complement resistance (59,
62, 69), and changes in the outer membrane protein profile of
the dsbA mutant could account for similar effects in H. influ-
enzae. The roles of the other potential DsbA substrates in H.
influenzae (Table 2) have not been defined, and their putative
homologs in E. coli do not appear to mediate virulence-related
functions. Furthermore, the complete set of H. influenzae
DsbA substrates remains to be determined experimentally.
Investigation of the virulence properties conferred by proteins
that contain DsbA-dependent disulfide bonds in H. influenzae
will likely uncover important aspects of the pathogenesis of this
bacterium and may lead to novel approaches to treatment or
prevention of invasive disease.
We thank Sanjay Ram for assistance and advice and Jeffrey Gawron-
ski for helpful comments.
This work was supported in part by a grant from the American Heart
Association and by NIH NIAID grant 1RO1-AI49437 to B.J.A.
1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic local alignment search tool. J. Mol. Biol. 215:403–410.
2. Ammendola, S., P. Pasquali, C. Pistoia, P. Petrucci, P. Petrarca, G.
Rotilio, and A. Battistoni. 2007. The high affinity Zn2?uptake system
ZnuABC is required for bacterial zinc homeostasis in intracellular envi-
ronments and contributes to virulence of Salmonella enterica. Infect.
3. Andersen, C. L., A. Matthey-Dupraz, D. Missiakas, and S. Raina. 1997. A
new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in
disulphide bond formation, required for recycling DsbA/DsbB and DsbC
redox proteins. Mol. Microbiol. 26:121–132.
4. Anderson, M. E., and A. Meister. 1980. Dynamic state of glutathione in
blood plasma. J. Biol. Chem. 255:9530–9533.
5. Anderson, P., R. B. Johnston, Jr., and D. H. Smith. 1972. Human serum
activities against Haemophilus influenzae, type b. J. Clin. Investig. 51:
6. Ausubel, F. M., R. Brent, R. E. Kingston, D. E. Moore, J. G. Seidman, J. A.
Smith, and K. Struhl (ed.). 1995. Current protocols in molecular biology.
John Wiley & Sons, Inc., New York, NY.
7. Barcak, G. J., M. S. Chandler, R. J. Redfield, and J. F. Tomb. 1991. Genetic
systems in Haemophilus influenzae. Methods Enzymol. 204:321–342.
8. Bardwell, J. C., J. O. Lee, G. Jander, N. Martin, D. Belin, and J. Beckwith.
1993. A pathway for disulfide bond formation in vivo. Proc. Natl. Acad. Sci.
9. Bardwell, J. C. A., K. McGovern, and J. Beckwith. 1991. Identification of a
protein required for disulfide bond formation in vivo. Cell 76:581–589.
10. Bessette, P. H., J. J. Cotto, H. F. Gilbert, and G. Georgiou. 1999. In vivo and
in vitro function of the Escherichia coli periplasmic cysteine oxidoreductase
DsbG. J. Biol. Chem. 274:7784–7792.
11. Bookwalter, J. E., J. A. Jurcisek, S. D. Gray-Owen, S. Fernandez, G. McGillivary,
and L. O. Bakaletz. 2008. A CEACAM1 homologue plays a pivotal role in non-
typeable Haemophilus influenzae colonization of the chinchilla nasopharynx via the
OMP P5-homologous adhesin. Infect. Immun. 76:48–55.
12. Bringer, M. A., N. Rolhion, A. L. Glasser, and A. Darfeuille-Michaud. 2007.
The oxidoreductase DsbA plays a key role in the ability of the Crohn’s
disease-associated adherent-invasive Escherichia coli strain LF82 to resist
macrophage killing. J. Bacteriol. 189:4860–4871.
13. CDC. 2002. Progress toward elimination of Haemophilus influenzae type b
invasive disease among infants and children—United States, 1998–2000.
MMWR Morb. Mortal. Wkly. Rep. 51:234–237.
14. Cope, L. D., R. Yogev, U. Muller-Eberhard, and E. J. Hansen. 1995. A gene
cluster involved in the utilization of both free heme and heme:hemopexin by
Haemophilus influenzae type b. J. Bacteriol. 177:2644–2653.
15. Cuthill, S. L., M. M. Farley, and L. G. Donowitz. 1999. Nontypable Hae-
mophilus influenzae meningitis. Pediatr. Infect. Dis. J. 18:660–662.
16. Daines, D. A., L. A. Cohn, H. N. Coleman, K. S. Kim, and A. L. Smith. 2003.
Haemophilus influenzae Rd KW20 has virulence properties. J. Med. Micro-
17. D’Mello, R. A., P. R. Langford, and J. S. Kroll. 1997. Role of bacterial
Mn-cofactored superoxide dismutase in oxidative stress responses, nasopha-
ryngeal colonization, and sustained bacteremia caused by Haemophilus in-
fluenzae type b. Infect. Immun. 65:2700–2706.
18. Erwin, A. L., K. L. Nelson, T. Mhlanga-Mutangadura, P. J. Bonthuis, J. L.
1506 ROSADINI ET AL.INFECT. IMMUN.
Geelhood, G. Morlin, W. C. Unrath, J. Campos, D. W. Crook, M. M. Farley,
F. W. Henderson, R. F. Jacobs, K. Muhlemann, S. W. Satola, L. van Alphen,
M. Golomb, and A. L. Smith. 2005. Characterization of genetic and pheno-
typic diversity of invasive nontypeable Haemophilus influenzae. Infect. Im-
19. Figueira, M. A., S. Ram, R. Goldstein, D. W. Hood, E. R. Moxon, and S. I.
Pelton. 2007. Role of complement in defense of the middle ear revealed by
restoring the virulence of nontypeable Haemophilus influenzae siaB mutants.
Infect. Immun. 75:325–333.
20. Fleischmann, R. D., M. D. Adams, O. White, R. A. Clayton, E. F. Kirkness,
A. R. Kerlavage, C. J. Bult, J. F. Tomb, B. A. Dougherty, J. M. Merrick, et
al. 1995. Whole-genome random sequencing and assembly of Haemophilus
influenzae Rd. Science 269:496–512.
21. Frech, C., M. Wunderlich, R. Glockshuber, and F. X. Schmid. 1996. Pref-
erential binding of an unfolded protein to DsbA. EMBO J. 15:392–398.
22. Gilligan, P. H. 1991. Microbiology of airway disease in patients with cystic
fibrosis. Clin. Microbiol. Rev. 4:35–51.
23. Gonzalez, M. D., C. A. Lichtensteiger, and E. R. Vimr. 2001. Adaptation of
signature-tagged mutagenesis to Escherichia coli K1 and the infant-rat model
of invasive disease. FEMS Microbiol. Lett. 198:125–128.
24. Granick, S., and H. Gilder. 1946. The porphyrin requirements of Haemophi-
lus influenzae and some functions of the vinyl and propionic acid side chains
of heme. J. Gen. Physiol. 30:1–13.
25. Griffin, R., C. D. Bayliss, M. A. Herbert, A. D. Cox, K. Makepeace, J. C.
Richards, D. W. Hood, and E. R. Moxon. 2005. Digalactoside expression in
the lipopolysaccharide of Haemophilus influenzae and its role in intravascular
survival. Infect. Immun. 73:7022–7026.
26. Ha, U. H., Y. Wang, and S. Jin. 2003. DsbA of Pseudomonas aeruginosa is
essential for multiple virulence factors. Infect. Immun. 71:1590–1595.
27. Hanson, M. S., C. Slaughter, and E. J. Hansen. 1992. The hbpA gene of
Haemophilus influenzae type b encodes a heme-binding lipoprotein con-
served among heme-dependent Haemophilus species. Infect. Immun. 60:
28. Herbert, M. A., S. Hayes, M. E. Deadman, C. M. Tang, D. W. Hood, and E. R.
Moxon. 2002. Signature tagged mutagenesis of Haemophilus influenzae iden-
tifies genes required for in vivo survival. Microb. Pathog. 33:211–223.
29. Hiniker, A., and J. C. Bardwell. 2004. In vivo substrate specificity of periplas-
mic disulfide oxidoreductases. J. Biol. Chem. 279:12967–12973.
30. Ho, D. K., S. Ram, K. L. Nelson, P. J. Bonthuis, and A. L. Smith. 2007. lgtC
expression modulates resistance to C4b deposition on an invasive nontype-
able Haemophilus influenzae. J. Immunol. 178:1002–1012.
31. Hood, D. W., M. E. Deadman, A. D. Cox, K. Makepeace, A. Martin, J. C.
Richards, and E. R. Moxon. 2004. Three genes, lgtF, lic2C and lpsA, have a
primary role in determining the pattern of oligosaccharide extension from
the inner core of Haemophilus influenzae LPS. Microbiology 150:2089–2097.
32. Jackson, M. W., and G. V. Plano. 1999. DsbA is required for stable expres-
sion of outer membrane protein YscC and for efficient Yop secretion in
Yersinia pestis. J. Bacteriol. 181:5126–5130.
33. Jacob-Dubuisson, F., J. Pinkner, Z. Xu, R. Striker, A. Padmanhaban, and
S. J. Hultgren. 1994. PapD chaperone function in pilus biogenesis depends
on oxidant and chaperone-like activities of DsbA. Proc. Natl. Acad. Sci. USA
34. Kadokura, H., F. Katzen, and J. Beckwith. 2003. Protein disulfide bond
formation in prokaryotes. Annu. Rev. Biochem. 72:111–135.
35. Kadokura, H., H. Tian, T. Zander, J. C. Bardwell, and J. Beckwith. 2004.
Snapshots of DsbA in action: detection of proteins in the process of oxidative
folding. Science 303:534–537.
36. Kim, S., K. Watanabe, T. Shirahata, and M. Watarai. 2004. Zinc uptake
system (znuA locus) of Brucella abortus is essential for intracellular survival
and virulence in mice. J. Vet. Med. Sci. 66:1059–1063.
37. Klein, J. O. 1997. Role of nontypeable Haemophilus influenzae in pediatric
respiratory tract infections. Pediatr. Infect. Dis. J. 16:S5–8.
38. Krupp, R., C. Chan, and D. Missiakas. 2001. DsbD-catalyzed transport of
electrons across the membrane of Escherichia coli. J. Biol. Chem. 276:3696–
39. Leichert, L. I., and U. Jakob. 2004. Protein thiol modifications visualized in
vivo. PLoS Biol. 2:e333.
40. Leroy, M., H. Cabral, M. Figueira, V. Bouchet, H. Huot, S. Ram, S. I. Pelton,
and R. Goldstein. 2007. Multiple consecutive lavage samplings reveal greater
burden of disease and provide direct access to the nontypeable Haemophilus
influenzae biofilm in experimental otitis media. Infect. Immun. 75:4158–4172.
41. Lu, D., B. Boyd, and C. A. Lingwood. 1997. Identification of the key protein
for zinc uptake in Hemophilus influenzae. J. Biol. Chem. 272:29033–29038.
42. Miki, T., N. Okada, and H. Danbara. 2004. Two periplasmic disulfide oxi-
doreductases, DsbA and SrgA, target outer membrane protein SpiA, a com-
ponent of the Salmonella pathogenicity island 2 type III secretion system.
J. Biol. Chem. 279:34631–34642.
43. Missiakas, D., C. Georgopoulos, and S. Raina. 1993. Identification and
characterization of the Escherichia coli gene dsbB, whose product is involved
in the formation of disulfide bonds in vivo. Proc. Natl. Acad. Sci. USA
44. Moller, L. V., A. G. Regelink, H. Grasselier, J. E. Dankert-Roelse, J.
Dankert, and L. van Alphen. 1995. Multiple Haemophilus influenzae strains
and strain variants coexist in the respiratory tract of patients with cystic
fibrosis. J. Infect. Dis. 172:1388–1392.
45. Morton, D. J., L. L. Madore, A. Smith, T. M. Vanwagoner, T. W. Seale, P. W.
Whitby, and T. L. Stull. 2005. The heme-binding lipoprotein (HbpA) of
Haemophilus influenzae: role in heme utilization. FEMS Microbiol. Lett.
46. Morton, D. J., A. Smith, Z. Ren, L. L. Madore, T. M. VanWagoner, T. W.
Seale, P. W. Whitby, and T. L. Stull. 2004. Identification of a haem-utiliza-
tion protein (Hup) in Haemophilus influenzae. Microbiology 150:3923–3933.
47. Morton, D. J., A. Smith, T. M. VanWagoner, T. W. Seale, P. W. Whitby, and
T. L. Stull. 2007. Lipoprotein e (P4) of Haemophilus influenzae: role in heme
utilization and pathogenesis. Microbes Infect. 9:932–939.
48. Moxon, E. R., and T. F. Murphy. 2000. Haemophilus influenzae, p. 2369–
2378. In G. L. Mandell, J. R. Bennett, and R. Dolin (ed.), Mandell, Douglas,
and Bennett’s principles and practices of infectious diseases, 5th ed., vol. 2.
Churchill Livingstone, New York, NY.
49. Murphy, T. F., A. L. Brauer, A. T. Schiffmacher, and S. Sethi. 2004. Persis-
tent colonization by Haemophilus influenzae in chronic obstructive pulmo-
nary disease. Am. J. Respir. Crit. Care Med. 170:266–272.
50. Murphy, T. F., and S. Sethi. 2002. Chronic obstructive pulmonary disease:
role of bacteria and guide to antibacterial selection in the older patient.
Drugs Aging 19:761–775.
51. Nakamoto, H., and J. C. Bardwell. 2004. Catalysis of disulfide bond forma-
tion and isomerization in the Escherichia coli periplasm. Biochim. Biophys.
52. Nickitenko, A. V., S. Trakhanov, and F. A. Quiocho. 1995. 2 A resolution
structure of DppA, a periplasmic dipeptide transport/chemosensory recep-
tor. Biochemistry 34:16585–16595.
53. Nizet, V., K. F. Colina, J. R. Almquist, C. E. Rubens, and A. L. Smith. 1996.
A virulent nonencapsulated Haemophilus influenzae. J. Infect. Dis. 173:180–
54. O’Neill, J. M., J. W. St. Geme III, D. Cutter, E. E. Adderson, J. Anyanwu,
R. F. Jacobs, and G. E. Schutze. 2003. Invasive disease due to nontypeable
Haemophilus influenzae among children in Arkansas. J. Clin. Microbiol.
55. Peek, J. A., and R. K. Taylor. 1992. Characterization of a periplasmic thiol:
disulfide interchange protein required for the functional maturation of se-
creted virulence factors of Vibrio cholerae. Proc. Natl. Acad. Sci. USA 89:
56. Ram, S., A. D. Cox, J. C. Wright, U. Vogel, S. Getzlaff, R. Boden, J. Li, J. S.
Plested, S. Meri, S. Gulati, D. C. Stein, J. C. Richards, E. R. Moxon, and
P. A. Rice. 2003. Neisserial lipooligosaccharide is a target for complement
component C4b. Inner core phosphoethanolamine residues define C4b link-
age specificity. J. Biol. Chem. 278:50853–50862.
57. Ram, S., J. Ngampasutadol, A. D. Cox, A. M. Blom, L. A. Lewis, F. St.
Michael, J. Stupak, S. Gulati, and P. A. Rice. 2007. Heptose I glycan sub-
stitutions on Neisseria gonorrhoeae lipooligosaccharide influence C4b-bind-
ing protein binding and serum resistance. Infect. Immun. 75:4071–4081.
58. Rietsch, A., P. Bessette, G. Georgiou, and J. Beckwith. 1997. Reduction of
the periplasmic disulfide bond isomerase, DsbC, occurs by passage of elec-
trons from cytoplasmic thioredoxin. J. Bacteriol. 179:6602–6608.
59. Ristow, P., P. Bourhy, F. W. McBride, C. P. Figueira, M. Huerre, P. Ave, I. S.
Girons, A. I. Ko, and M. Picardeau. 2007. The OmpA-like protein Loa22 is
essential for leptospiral virulence. PLoS Pathog. 3:e97.
60. Rubin, L. G., and J. W. St. Geme III. 1993. Role of lipooligosaccharide in
virulence of the Brazilian purpuric fever clone of Haemophilus influenzae
biogroup aegyptius for infant rats. Infect. Immun. 61:650–655.
61. Seale, T. W., D. J. Morton, P. W. Whitby, R. Wolf, S. D. Kosanke, T. M.
VanWagoner, and T. L. Stull. 2006. Complex role of hemoglobin and
hemoglobin-haptoglobin binding proteins in Haemophilus influenzae vir-
ulence in the infant rat model of invasive infection. Infect. Immun.
62. Serino, L., B. Nesta, R. Leuzzi, M. R. Fontana, E. Monaci, B. T. Mocca, E.
Cartocci, V. Masignani, A. E. Jerse, R. Rappuoli, and M. Pizza. 2007.
Identification of a new OmpA-like protein in Neisseria gonorrhoeae involved
in the binding to human epithelial cells and in vivo colonization. Mol.
63. Sethi, S., and T. F. Murphy. 2001. Bacterial infection in chronic obstructive
pulmonary disease in 2000: a state-of-the-art review. Clin. Microbiol. Rev.
64. Sone, M., Y. Akiyama, and K. Ito. 1997. Differential in vivo roles played by
DsbA and DsbC in the formation of protein disulfide bonds. J. Biol. Chem.
65. Stenson, T. H., and A. A. Weiss. 2002. DsbA and DsbC are required for
secretion of pertussis toxin by Bordetella pertussis. Infect. Immun. 70:2297–
66. Tinsley, C. R., R. Voulhoux, J. L. Beretti, J. Tommassen, and X. Nassif.
2004. Three homologues, including two membrane-bound proteins, of the
disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacte-
rial growth and biogenesis of functional type IV pili. J. Biol. Chem.
VOL. 76, 2008DsbA AND H. INFLUENZAE PATHOGENESIS 1507
67. Tomb, J. F. 1992. A periplasmic protein disulfide oxidoreductase is required Download full-text
for transformation of Haemophilus influenzae Rd. Proc. Natl. Acad. Sci. USA
68. Watarai, M., T. Tobe, M. Yoshikawa, and C. Sasakawa. 1995. Disulfide
oxidoreductase activity of Shigella flexneri is required for release of Ipa
proteins and invasion of epithelial cells. Proc. Natl. Acad. Sci. USA 92:4927–
69. Weiser, J. N., and E. C. Gotschlich. 1991. Outer membrane protein A
(OmpA) contributes to serum resistance and pathogenicity of Escherichia
coli K-1. Infect. Immun. 59:2252–2258.
70. White, D. C., and S. Granick. 1963. Hemin Biosynthesis in Haemophilus. J.
71. Wong, S. M., and B. J. Akerley. 2003. Inducible expression system and
marker-linked mutagenesis approach for functional genomics of Haemophi-
lus influenzae. Gene 316:177–186.
72. Wong, S. M., K. R. Alugupalli, S. Ram, and B. J. Akerley. 2007. The ArcA
regulon and oxidative stress resistance in Haemophilus influenzae. Mol. Mi-
73. Yang, X., T. Becker, N. Walters, and D. W. Pascual. 2006. Deletion of znuA
virulence factor attenuates Brucella abortus and confers protection against
wild-type challenge. Infect. Immun. 74:3874–3879.
74. Yu, J., H. Webb, and T. R. Hirst. 1992. A homologue of the Escherichia coli
DsbA protein involved in disulphide bond formation is required for entero-
toxin biogenesis in Vibrio cholerae. Mol. Microbiol. 6:1949–1958.
75. Zapun, A., and T. E. Creighton. 1994. Effects of DsbA on the disulfide
folding of bovine pancreatic trypsin inhibitor and alpha-lactalbumin. Bio-
76. Zapun, A., D. Missiakas, S. Raina, and T. E. Creighton. 1995. Structural and
functional characterization of DsbC, a protein involved in disulfide bond
formation in Escherichia coli. Biochemistry 34:5075–5089.
77. Zhang, H. Z., and M. S. Donnenberg. 1996. DsbA is required for stability of
the type IV pilin of enteropathogenic Escherichia coli. Mol. Microbiol. 21:
Editor: J. B. Bliska
1508 ROSADINI ET AL.INFECT. IMMUN.