INFECTION AND IMMUNITY, Sept. 2009, p. 3969–3977
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 9
Role of the Type III Secretion System in a Hypervirulent Lineage of
Anne M. Buboltz,1,2Tracy L. Nicholson,3Laura S. Weyrich,1,2and Eric T. Harvill1*
Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, 115 Henning Building, University Park,
Pennsylvania 168021; Graduate Program in Biochemistry, Microbiology, and Molecular Biology, The Pennsylvania State University,
108 Althouse Building, University Park, Pennsylvania 168022; and Respiratory Diseases of Livestock Research Unit,
National Animal Disease Center, Agricultural Research Service, USDA, Ames, Iowa3
Received 6 November 2008/Returned for modification 16 December 2008/Accepted 2 July 2009
Despite the fact that closely related bacteria can cause different levels of disease, the genetic changes that
cause some isolates to be more pathogenic than others are generally not well understood. We use a combination
of approaches to determine which factors contribute to the increased virulence of a Bordetella bronchiseptica
lineage. A strain isolated from a host with B. bronchiseptica-induced disease, strain 1289, was 60-fold more
virulent in mice than one isolated from an asymptomatically infected host, strain RB50. Transcriptome
analysis and quantitative reverse transcription-PCR showed that the type III secretion system (TTSS) genes
were more highly expressed by strain 1289 than strain RB50. Compared to strain RB50, strain 1289 exhibited
greater TTSS-mediated cytotoxicity of a mammalian cell line. Additionally, we show that the increase in
virulence of strain 1289 compared to that of RB50 was partially attributable to the TTSS. Using multilocus
sequence typing, we identified another strain from the same lineage as strain 1289. Similar to strain 1289, we
implicate the TTSS in the increased virulence of this strain. Together, our data suggest that the TTSS is
involved in the increased virulence of a B. bronchiseptica lineage which appears to be disproportionately
associated with disease. These data are consistent with the view that B. bronchiseptica lineages can have
different levels of virulence, which may contribute to this species’ ability to cause different severities of
Although the disease caused by different strains of patho-
genic bacteria is known to vary, the molecular basis for these
differences has been difficult to disentangle from the many
other genetic changes that occur as strains diverge. Recently, a
growing number of studies have identified factors that contrib-
ute to increased virulence of bacterial lineages by using a
combination of genome-wide analyses, phylogenetics, muta-
tional analysis, and host infection models (15, 46, 49–51, 56,
58). Horizontal gene transfer of novel virulence factors, phage
integration, phenotypic variation, gene loss, and mutation have
been shown to alter the phenotype or severity of disease (15,
Bordetella bronchiseptica is a gram-negative respiratory
pathogen that infects a wide range of mammals and is closely
related to Bordetella pertussis and Bordetella parapertussis, the
causative agents of whooping cough in humans (18, 33, 36).
Colonization of hosts by B. bronchiseptica can lead to a range
of diseases, from lethal pneumonia to asymptomatic infection
(18), which is thought to be caused by differences in host
immune status, polymicrobial infection, and/or bacterial strain
variation (18, 33). However, in inbred and specific-pathogen-
free mice, the 50% lethal dose (LD50) can still differ by up to
100,000-fold between bacterial strains, suggesting that substan-
tial differences in virulence may be due to strain variation
alone (5, 19, 20).
While the population structure of these bacteria appears to
be clonal, B. pertussis and B. parapertussis are more monomor-
phic than B. bronchiseptica strains, and isolates of this species
can be related more distantly to each other than to either of
the human-associated pathogens (11, 36, 37, 60). Previously, it
was shown that differences in gene regulation between B. bron-
chiseptica strains can correlate with phylogenetic lineage (17)
and strains can differ in virulence factor expression (3, 19, 31,
36, 47). Recently, we showed that phylogenetic lineages can
differ in virulence factor expression and virulence, as a lineage
of B. bronchiseptica was found to lack expression of adenylate
cyclase toxin and exhibit decreased virulence (5).
B. bronchiseptica strains express many virulence factors, in-
cluding adhesins, secretion systems, autotransporters, and tox-
ins, that are globally regulated by the BvgAS two-component
signal transduction system (7, 53). In the nonvirulent, or Bvg?
phase, which occurs at 25°C or in the presence of chemical
modulators such as MgSO4or nicotinic acid, BvgAS is unable
to activate virulence-associated genes (10, 35, 38). During the
virulent, or Bvg?phase, which occurs when the bacteria are
grown at 37°C and in the absence of chemical modulators,
BvgAS activates the expression of a large set of virulence
factors (7, 10, 38). The Bvg?phase is both necessary and
sufficient for colonization of the respiratory tract (1, 8). Among
the Bvg-regulated genes are those encoding a type III secretion
system (TTSS) which is similar to others shown to directly
translocate effector proteins through a needle-like injection
apparatus directly into eukaryotic cells, disrupt host cell sig-
naling, and induce necrotic-like cell death (27, 62). Under
* Corresponding author. Mailing address: Department of Veteri-
nary and Biomedical Sciences, The Pennsylvania State University, 115
Henning Building, University Park, PA 16802. Phone: (814) 863-8522.
Fax: (814) 863-6140. E-mail: email@example.com.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 13 July 2009.
Bvg?conditions, the btr regulatory locus (including btrS, btrU,
btrW, and btrV) is transcribed (34). BtrS is an ECF sigma factor
that is necessary and sufficient for activating the more than 20
TTSS-related genes (bsc, bop, bsp, and bte) (34). BscN is the
putative ATPase that provides energy for the secretion of ef-
fector proteins and is required for the function of the TTSS
apparatus (62). TTSS gene products BopB, BopC, and BteA
have been shown to be secreted and required for cytotoxicity in
mammalian cells (28, 29, 41). In a murine model of infection,
the TTSS increases host interleukin-10 production and en-
hances persistence of B. bronchiseptica in the lower respiratory
tract (44, 61, 62).
While correlations between the level of virulence or disease
caused by B. bronchiseptica strains and specific bacterial factors
have been made (5, 39, 47), limited studies have directly tested
whether these factors cause some strains to be more virulent
than others (4) and whether these characteristics are associ-
ated with a particular phylogenetic lineage. Here, we use ge-
nome-wide analyses, phylogenetics, allelic exchange, and a mu-
rine model of infection to determine the bacterial factors that
contribute to the increased virulence of a B. bronchiseptica
lineage. We compared the relative virulence, as measured by
LD50, of B. bronchiseptica strains from a diseased (strain 1289)
or asymptomatically infected (strain RB50) host (8). Strain
1289 was approximately 60-fold more virulent than strain
RB50. Transcriptome analysis showed that TTSS-related genes
were more highly expressed by strain 1289 than RB50. The
TTSS-mediated cytotoxicity and virulence of 1289 was greater
than that of strain RB50. Using multilocus sequence typing
(MLST) analysis, we identified another strain from the same
sequence type (ST) as strain 1289 and showed that, similar to
strain 1289, the increased virulence of this strain was partially
attributable to the TTSS. Together, our data indicate that the
TTSS is involved in the increased virulence of a B. bronchisep-
tica lineage. This is consistent with the idea that different
phylogenetic lineages can differentially regulate their virulence
factors to modulate their overall level of virulence, which may
contribute to the ability of B. bronchiseptica strains to cause
different severities of respiratory disease.
MATERIALS AND METHODS
Bacterial strains and growth. B. bronchiseptica isolates, sources, locations,
dates, anatomical sites of isolation, references, and all available health and
necropsy reports (strains 1289, S308, and S314) are included herein or in Table
S1 in the supplemental material or have been previously described (strain RB50)
(8). In cases where there was an available health report, strains were grouped as
those from diseased hosts (i.e., having lower respiratory tract infection requiring
medical treatment or causing death) or strains isolated from a nondiseased host
(i.e., having an asymptomatic infection) (see Table S1 in the supplemental
material). For the rest of the isolates studied here, a more detailed clinical report
was not available. The effect of in vitro passaging on B. bronchiseptica strains has
been kept to a minimum, as all the isolates used in our murine model of infection
in this study have been minimally passaged (estimated to be less than five times).
All strains were maintained on Bordet-Gengou (BG) agar (Difco, Sparks, MD)
containing 10% sheep’s blood (Hema Resources, Aurora, OR) with 20 ?g/ml
streptomycin (Sigma, St. Louis, MO). For inoculation, bacteria were grown
overnight at 37°C in Stainer-Scholte (SS) broth (51a) to logarithmic phase,
bacterial density was measured by optical density read at 600 nm (OD600), and
bacteria were diluted in sterile phosphate-buffered saline (Omnipur, Gibbstown,
NJ) to the appropriate concentration. Inocula were confirmed by plating dilu-
tions on BG agar and counting the resulting colonies after 2 days of incubation
at 37°C, as previously described (22, 26). Because the Bvg?phase is required for
colonization of the respiratory tract, only strains that appeared Bvg?(domed and
?-hemolytic) were used in the murine model of infection. Strains that exhibited
rough colony morphology and lacked ?-hemolysis, an indication of avirulent
Bvg?mutants which commonly occur upon in vitro passaging, were excluded
from this type of analysis (52). For this reason, strains 1973 and 1987 were not
analyzed in vivo.
Animal experiments. Four- to 6-week-old C57BL/6 mice were obtained from
Jackson Laboratories at The Pennsylvania State University. All experiments
were completed in accordance with institutional guidelines. For inoculation,
mice were lightly sedated with 5% isofluorane (IsoFlo; Abbott Laboratories) in
oxygen and the indicated number of CFU of the appropriate B. bronchiseptica
strain in 50 ?l of phosphate-buffered saline was gently pipetted onto the external
nares as previously described (22, 26). For survival curves, groups of three or four
mice were inoculated with the indicated dose and percent survival was monitored
over a 28-day period. Mice with lethal bordetellosis, indicated by ruffled fur,
labored breathing, and diminished responsiveness, were euthanized to alleviate
unnecessary suffering (22, 32, 43). Statistical significance was calculated using a
Fisher’s exact test where groups of mice were compared in terms of survival or
death at a similar dose of two different strains, as previously described (5). To
quantify the number of bacteria in the lungs, trachea, and nasal cavity, groups of
three or four mice were inoculated with a sublethal dose (1 ? 104CFU) and
sacrificed at the time points indicated below. Bacterial numbers in all respiratory
organs were quantified as previously described (22, 25). The mean ? standard
error of the results was determined for each treatment group. Statistical signif-
icance in bacterial load between strains was calculated by using analysis of
covariance in Minitab (version 13.30; Minitab., Inc.). The explanatory variable
used was the bacterial strain, and a covariate for day was fitted to control for the
change in load over time (5). We report F values, the test statistic for analyses of
covariance, as well as P values, for full statistical disclosure (40). A P value of
?0.05 was taken as statistically significant. All animal experiments were repeated
at least twice with similar results.
Expression arrays and statistical analysis. The expression array was carried
out as previously described (5, 38). Briefly, bacteria were grown in SS broth,
subcultured at a starting OD600of 0.02 into 50 ml of SS broth, grown at 37°C for
24 h with shaking, and harvested in log phase (OD600of 1.0). Total RNA was
extracted with Trizol (Invitrogen, Carlsbad, CA), treated with RNase-free
DNase I (Invitrogen, Carlsbad, CA), and purified using RNeasy columns
(Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA was
isolated from two independent biological replicates of strains RB50 and 1289. A
two-color hybridization format was used, and dye swap experiments were per-
formed. For each reaction mixture, 5 ?g of cDNA was fluorescently labeled. The
two differentially labeled reaction mixtures to be compared were combined and
hybridized to a B. bronchiseptica strain RB50-specific long-oligonucleotide mi-
croarray (5, 38). The slides were then scanned using a GenePix 4000B microarray
scanner and analyzed with GenePix Pro software (Axon Instruments, Union City,
CA). The spots were assessed visually to identify those of low quality, and the
arrays were normalized so that the median of the ratio across each array was
equal to 1.0. Spots of low quality were identified and were filtered out prior to
analysis. Ratio data from the two biological replicates were compiled and nor-
malized based on the total Cy3 percent intensity and Cy5 percent intensity to
eliminate slide-to-slide variation. Gene expression data were then normalized to
the expression of 16S rRNA. The statistical significance of the gene expression
changes observed was assessed by using the significance analysis of microarrays
(SAM) program (59). A one-class unpaired SAM analysis using a false discovery
rate of 0.30% (?0.1%) was performed. All microarray expression data are
available in Table S2 in the supplemental material.
qRT-PCR. Quantitative reverse transcription-PCR (qRT-PCR) was com-
pleted as previously described (5, 38), and RNA was extracted as described for
the microarray experiment. One microgram of RNA from each biological rep-
licate was reverse transcribed using 300 ng of random oligonucleotide hexamers
and SuperScript III RTase (Invitrogen, Carlsbad, CA). The resulting cDNA was
diluted 1:1,000, and 1-?l amounts were used in qRT-PCR mixtures containing
300 nM primers that were designed with Primer Express software (Applied
Biosystems, Foster City, CA) and 2? SYBR green PCR master mix (Applied
Biosystems, Foster City, CA). To confirm the lack of DNA contamination,
reactions of mixtures without reverse transcriptase were completed. Dissociation
curve analysis was performed to verify product homogeneity. Threshold fluores-
cence was established within the geometric phase of exponential amplification,
and the cycle threshold (CT) determined for each reaction mixture. The CTfrom
all biological replicates for each strain was compiled, and the 16S RNA amplicon
was used as an internal control for data normalization. The change in transcript
level was determined by using the relative quantitative CTmethod (??CT) (48).
All primer sequences and changes in gene expression analyzed by qRT-PCR are
available (see Table S2 in the supplemental material).
3970BUBOLTZ ET AL.INFECT. IMMUN.
CGH and statistical analysis. Comparative genomic hybridization (CGH)
analysis was completed as previously described (5). Briefly, strains RB50 and
1289 were grown in SS broth at 37°C with shaking overnight and genomic DNA
was isolated from bacterial cultures by using a DNA extraction kit (Qiagen,
Valencia, CA) and digested with DpnII. For each labeling reaction, 2 ?g of
digested genomic DNA was randomly primed using Cy5 and Cy3 dye-labeled
nucleotides with BioPrime DNA labeling kits (Invitrogen, Carlsbad, CA), and
the two differentially labeled reaction mixtures to be compared were combined
and hybridized to a B. bronchiseptica RB50-specific long-oligonucleotide mi-
croarray (5, 38). Dye swap experiments were performed. Statistical analysis of
CGH data was performed with SAS software version 9.1.3 (SAS Institute, Inc.,
Cary, NC). A MODECLUS procedure (PROC MODECLUS) based on non-
parametric density estimation was used to cluster genes into divergent or non-
divergent groups. All CGH data are available in Table S3 in the supplemental
Deletion of bscN in B. bronchiseptica. The gene encoding the ATPase that
provides energy for secretion of proteins via the TTSS, bscN, was deleted from
strain RB50 as previously described (62). The deletion of bscN from strains 1289
and S308 was completed as follows. The 419 base pairs upstream and the first
three codons of the bscN gene were PCR amplified using primers flanked with
EcoRI on the 5? end and BamHI on the 3? end (F-ATCGAATTCCGGATCA
GGCGGAGAAGA and R-TAAGGATCCCTGACGCATGCCCCTATC, re-
spectively). The 420 base pairs downstream and the last three codons of the bscN
gene were PCR amplified using primers flanked with BamHI on the 5? end and
EcoRI on the 3? end (F-CGCGGATCCGAATCCTAATGGACCTGG and R-
TAGGAATTCTCCAGGCTCTCGCGCAAG, respectively). The PCR condi-
tions were 95°C for 5 min; 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for
1 min; and 72°C for 5 min. These fragments were PCR purified (Qiagen, Va-
lencia, CA), BamHI digested (New England Biolabs), gel purified (Qiagen,
Valencia, CA), and ligated overnight at 4°C (New England Biolabs). The ligation
product was then amplified with the 5? F and 3? R primers as described above.
The 846-bp product was ligated into the TOPO-TA vector and transformed into
Mach1 DH5? cells (Invitrogen, Carlsbad, CA). The presence of the insert in
TOPO-TA was screened for by the loss of ?-galactosidase activity and EcoRI
digestion of the plasmid from resulting transformants. The 838-bp insert was
digested from TOPO-TA, gel purified, and ligated overnight into the EcoRI-
digested pSS4245, a new Bordetella allelic exchange vector (S. Stibitz, unpub-
lished data). The ligation product was transformed as described above. The
presence of the insert in pSS4245 was screened for by extracting the plasmid
from the resulting transformants and digesting it with EcoRI. The resulting
positive clone was named pSS4245?bscN. The positive clones were sequenced
after insertion into TOPO-TA and pSS4245 to ensure that PCR-induced muta-
tions did not occur. DH5? harboring pSS4545?bscN or a plasmid competent for
mating, pSS1827 (54), and the appropriate B. bronchiseptica strain grown under
Bvg?conditions by growth on BG plus 50 mM MgSO4was mated for 4 h on a
BG–10 mM MgCl2–50 mM MgSO4plate at 37°C. Then, B. bronchiseptica con-
taining pSS4245?bscN was positively selected for by using BG-streptomycin-
kanamycin–50 mM MgSO4plates and incubated for 5 days at 37°C. The resulting
B. bronchiseptica colonies were streaked onto BG-streptomycin-kanamycin–50
mM MgSO4and grown at 37°C for 4 days. The resulting colonies were streaked
onto BG plates and incubated for 2 days at 37°C, which resulted in colonies
lacking pSS4245 and containing either the wild-type or knockout gene. Colonies
were screened for the presence of either the wild-type or knockout gene by using
screening primers (F-ATCGACTACTTCGCGGGTATCGAGAA and R-GAG
CAGCTGGATTTCATGCTCGTG) which detected either the wild-type bscN
gene (2,003 bp) or the bscN deletion (678 bp) with PCR conditions of 95°C for
5 min; 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min; and 72°C for
5 min. To further confirm the presence and absence of the bscN gene, screening
primers which amplify the middle of the bscN gene and could therefore only
amplify the wild-type gene (1,071 bp) were used (F-GAACGATCATCAAGGC
CGTCGTTC and R-GTCCTGGTACTTGGCCATCAGTTC). The absence of
pSS4245 was confirmed by growth on BG-streptomycin plates and lack of growth
on BG-kanamycin plates.
Cytotoxicity assay. Cytotoxicity assays were carried out as previously described
(34, 62). Briefly, J774 macrophages were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin,
1% nonessential amino acids, and 1% sodium pyruvate to 85% confluence in 5%
CO2at 37°C. Then, warmed RPMI medium lacking phenol red and with 5% fetal
bovine serum, 1% L-glutamine, 1% nonessential amino acids, and 1% sodium
pyruvate was used to replace the Dulbecco’s modified Eagle’s medium. Bacterial
infections were carried out using a multiplicity of infection (MOI) of 10, and
bacterial suspensions were centrifuged onto the macrophage cells at 250 ? g for
5 min and incubated in 5% CO2at 37°C for the amount of time indicated below.
The cell culture supernatants were collected, and the percent lipodehydrogenase
(LDH) release was analyzed by using a Cytotox96 kit (Promega) according to the
manufacturer’s instructions. Statistical significance in percent cytotoxicity be-
tween strains was calculated by using a Tukey simultaneous test in Minitab
(version 13.30; Minitab, Inc.) (40). A P value of ?0.05 was taken as statistically
MLST and phylogenetic tree construction. MLST analysis was performed as
previously described (5, 11). In this study, the STs of three strains (S308, S314,
and 973) were determined (see Table S1 in the supplemental material). All
alleles were double-strand sequenced at The Pennsylvania State University’s
Sequencing Center. The sequences were trimmed, and alleles and STs were
designatedby using theBordetella
/bordetella) (5, 11, 23). Using MEGA 4.0, the alleles were concatenated and
aligned, and an unweighted pair group method with arithmetic mean tree with
1,000 bootstraps using the K2 model was constructed for these 3 strains and for
58 strains whose STs were previously determined (5, 11, 57) (see Table S1 in the
Accession numbers. All microarray expression data have been deposited in
MAIMExpress under the accession number E-MEXP-1736, and all CGH data
have been deposited in MIAMExpress under the accession number E-MEXP-
MLST database (http://pubmlst.org
B. bronchiseptica strain 1289 is more virulent than strain
RB50 in a mouse intranasal challenge model. Previous studies
have shown that B. bronchiseptica strains can vary widely in
virulence (5, 19, 20). To establish a system in which we could
measure the contribution of specific factors to the difference in
virulence between strains, we compared the LD50, a measure
of their virulence, of B. bronchiseptica strains in inbred, spe-
cific-pathogen-free mice. B. bronchiseptica strain RB50 was
isolated from the nasal cavity of an asymptomatically infected
host (8). Consistent with previous studies of this strain, inoc-
ulation with 1 ? 106, 3 ? 106, or 6 ? 106CFU of B. bronchi-
septica strain RB50 led to 100%, 50%, and 0% survival, re-
spectively (Fig. 1A) (5). B. bronchiseptica strain 1289 was
isolated from the thoracic cavity of a host with a lethal B.
bronchiseptica infection (see Table S1 in the supplemental ma-
terial). When inoculated with 1 ? 104, 5 ? 104, or 1 ? 105CFU
of strain 1289, 100%, 50%, and 0% of the mice survived the
infection, respectively (Fig. 1B), which indicates that the LD50
of strain 1289 is 60-fold lower, or 2.95 million CFU fewer, than
that of strain RB50. Although these two isolates did not differ
in growth rate in vitro (data not shown), we examined whether
the greater virulence of strain 1289 might allow it to colonize
the respiratory tract to a higher level than strain RB50. Groups
of 15 mice were inoculated with a sublethal dose (1 ? 104
CFU) of either strain RB50 or 1289, and respiratory organs
were excised to quantify bacterial loads on days 0, 3, 7, 14, and
28 postinoculation (Fig. 1C). In the lungs, strain RB50 peaked
at approximately 1 ? 105CFU and was reduced to below the
limit of detection (10 CFU) by day 28 postinoculation (Fig.
1C). The bacterial load of strain 1289 was approximately 10-
fold higher than that of strain RB50 over the course of infec-
tion (Fig. 1C) (for bacterial strain comparisons, F1.27? 7.03
and P ? 0.013). Even 24 h postinoculation, the bacterial load
of strain 1289 was 10-fold higher than that of strain RB50 (P ?
0.002), indicating that the numbers of strain 1289 are higher
than the numbers of strain RB50 early after infection (data not
shown). The bacterial load did not differ significantly between
strain RB50 and 1289 in the trachea or nasal cavity over the
course of infection (see Fig. S4 in the supplemental material).
Combined, these data indicate that strain 1289 is more virulent
VOL. 77, 2009 TTSS AND HYPERVIRULENCE OF A B. BRONCHISEPTICA LINEAGE3971
than strain RB50 and colonizes the lower respiratory tract at a
TTSS genes are upregulated in strain 1289. To identify
candidate bacterial genes that correlated with the increased
virulence of strain 1289, a comparison of whole-genome tran-
scriptome analyses was performed between strains 1289 and
RB50 (Fig. 2A). Of the 5,013 genes represented on the RB50-
specific microarray, 646 were downregulated in strain 1289
relative to their expression levels in strain RB50. These
included 49 transporter genes; 47 metabolism-related tran-
scripts; 51 transcriptional regulator genes; 27 electron trans-
porter genes; 11 two-component system genes; 7 transcrip-
tional or translational genes; 1 protein biosynthesis-related
transcript; 74 exported or membrane protein genes; 72 phage-
related transcripts; and 202 hypothetical, predicted, or proba-
ble genes. Additionally, five genes classified as virulence fac-
tors were downregulated two- to eightfold in strain 1289
compared to their levels of expression in strain RB50; these
were genes for filamentous hemagglutinin B (fhaB), filamen-
tous hemagglutinin S (fhaS), Bordetella colonization factor
(bcfA), Bordetella resistance to killing A (brkA), and one O-
antigen-related gene (wbmS) (Fig. 2A) (see Table S2 in the
supplemental material) (6, 9, 13, 24, 55). The downregulated
expression of brkA and fhaB in strain 1289 was confirmed by
qRT-PCR (see Table S2 in the supplemental material). When
CGH analysis was completed on these strains, none of the
known virulence factors were identified as divergent in strain
1289 (see Table S3 in the supplemental material), suggesting
that the decreased signal of these virulence factors in strain
1289 identified in our transcriptome analysis is due to down-
regulation rather than sequence divergence.
We were particularly interested in determining which genes
were upregulated in strain 1289, as they might contribute to the
greater virulence of this strain. Six hundred seven genes were
identified as upregulated in strain 1289 relative to their expres-
sion levels in strain RB50. These included 60 transporter
genes; 67 metabolism-related transcripts; 23 transcriptional
regulator genes; 42 electron transporter genes; 7 two-compo-
nent system genes; 16 transcriptional or translational genes; 40
protein biosynthesis-related transcripts; 75 exported or mem-
brane protein genes; 16 phage-related transcripts; and 154
hypothetical, predicted, or probable genes. Thirty-three genes
associated with known virulence factors were upregulated in
strain 1289 compared to their expression levels in strain RB50.
Four of these, the genes for cyclolysin-activating lysine-acyl-
transferase (cyaC), Bordetella resistance to killing B (brkB), an
O-antigen-related protein (wbmJ), and pertussis toxin subunit
4 precursor (ptxD), were upregulated by 1.8-fold or more in
strain 1289 (Fig. 2A) (see Table S2 in the supplemental mate-
rial). The remaining 29 virulence-associated genes are related
to the TTSS and were upregulated from 1.4- to 8.5-fold in
strain 1289 over their expression levels in strain RB50 (Fig. 2A
and B). As expected, there was a strong correlation between
expression levels analyzed by microarray and qRT-PCR results
(R ? 0.914) (see Table S2 in the supplemental material).
Although qRT-PCR did not confirm the upregulation of cyaC,
all the TTSS-related genes examined were upregulated 2.6- to
12.8-fold in strain 1289 compared to their levels of expression
in strain RB50 (Fig. 2C; see also Table S2 in the supplemental
material). The upregulation of these virulence factor genes in
strain 1289 does not appear to be due to gene duplication, as
no genes were identified as duplicated in CGH analysis (see
Table S3 in the supplemental material).
The TTSS is involved in the increased cytotoxicity and vir-
ulence of strain 1289. The increased expression of such a large
set of genes with a known coordinated function in virulence led
us to hypothesize that strain 1289 exhibited greater TTSS-
mediated effects than strain RB50. One well-described func-
tion attributed to the TTSS is cytotoxicity for a variety of
mammalian cells (14, 34, 62). Since nearly all TTSS-related
genes are upregulated in strain 1289, we hypothesized that this
strain may cause more TTSS-mediated cytotoxicity than strain
RB50. Although J774 macrophages treated with medium alone
did not release cytoplasmic LDH, infection of these macro-
phages with strain RB50 at an MOI of 10 caused 5%, 7%, 34%,
and 87% of their LDH to be released after 1, 2, 3, and 4 h of
FIG. 1. LD50s and quantification of lung bacterial loads of B. bron-
chiseptica strains RB50 and 1289. Groups of three or four C57BL/6
mice were inoculated intranasally with the indicated doses of strains
RB50 (A) or 1289 (B). Survival curves were generated by inoculating
mice with the indicated dose and determining the percent survival over
a 28-day period. (C) Groups of three mice were inoculated intranasally
with 1 ? 104CFU of strain RB50 or strain 1289. Bacterial loads in the
lungs were quantified 0, 3, 7, 14, and 28 days postinoculation. The
dashed line indicates the lower limit of detection. Bacterial numbers
are expressed as the log10means ? standard errors (error bars).
3972 BUBOLTZ ET AL.INFECT. IMMUN.
infection, respectively, indicating that RB50 is cytotoxic toward
macrophages (P ? 0.0001) (Fig. 3A), as previously reported
(34, 62). Upon infection with the same dose of strain 1289, the
percent LDH release was higher than that caused by strain
RB50 (P ? 0.0360), which indicates that strain 1289 causes
more rapid cytotoxicity of macrophages than strain RB50 (Fig.
3A). To determine if the cytotoxicity induced by these strains
is caused by the TTSS, isogenic mutants each lacking the bscN
gene (RB50?bscN and 1289?bscN) were used to compare
their cytotoxicity for J774 macrophages (Fig. 3A). The percent
LDH release caused by infection with RB50?bscN was lower
than that caused by its parental strain, RB50, and was not
significantly different from that of the medium control (P ?
0.0047 and P ? 0.5758, respectively), which confirms that the
TTSS of strain RB50 causes cytotoxicity toward macrophages
(Fig. 3A) (21, 62). Similarly, the percent LDH release caused
by infection with 1289?bscN was lower than that caused by its
wild-type counterpart and was not significantly different from
that in the medium control or RB50?bscN (P ? 0.0001, P ?
0.2960, and P ? 0.9857, respectively), the former of which
indicates that strain 1289 does not have a measurable TTSS-
independent mechanism of cytotoxicity (Fig. 3A). The greater
TTSS-dependent cytotoxicity caused by strain 1289 at earlier
time points suggests that strain 1289 causes more rapid TTSS-
mediated cytotoxicity than strain RB50.
Since the TTSS causes cytotoxicity in vitro and increases
bacterial numbers in vivo (21, 44), we hypothesized that the
TTSS contributes more to the virulence of strain 1289 than to
that of strain RB50. To test this, mice were inoculated with the
bscN deletion strains of RB50 and 1289 and the LD50s of these
FIG. 2. Whole-transcriptome and TTSS expression analysis of B. bronchiseptica strains RB50 and 1289. (A) Comparison of whole-transcrip-
tome analyses between strains RB50 and 1289. The x axis indicates the order of genes along the B. bronchiseptica strain RB50 5.3-megabase (Mb)
chromosome. The y axis indicates the change in expression level (as fold change in expression [FCE]) of each gene. Negative FCE values indicate
decreased gene expression of genes in strain 1289 compared to their levels of expression in strain RB50, and positive FCE values indicate increased
gene expression in strain 1289 compared to their levels in strain RB50. Genes of interest are labeled, with corresponding underscores. HP,
hypothetical protein gene; ?, phage-related gene; PEP, putative exported protein gene; BB4921, putative ferredoxin gene. (B and C) Comparison
of TTSS-related gene expression between strains RB50 and 1289 by microarray analysis (B) and qRT-PCR (C). The x axis indicates the genes
analyzed. The y axis indicates the FCE in strain 1289 over the expression level in strain RB50. Error bars represent the plus-or-minus standard
errors in panels A and B and the standard deviation in panel C.
FIG. 3. TTSS-mediated effect on cytotoxicity and virulence of B.
bronchiseptica strains RB50 and 1289. (A) Cytotoxicity in J774 macro-
phages treated with medium or infected with RB50, RB50?bscN, 1289,
or 1289?bscN for 1, 2, 3, and 4 h at an MOI of 10. The error bars
represent the plus-or-minus standard deviations. (B) Groups of three
or four C57BL/6 mice were inoculated intranasally with the indicated
doses of strains RB50?bscN or 1289?bscN. Survival curves were gen-
erated by inoculating mice with the indicated dose and determining the
percent survival over a 28-day period.
VOL. 77, 2009TTSS AND HYPERVIRULENCE OF A B. BRONCHISEPTICA LINEAGE 3973
isogenic mutant strains were determined. When inoculated
with 8.0 ? 106or 1.0 ? 107CFU of RB50?bscN, 100% and 0%
of the mice survived the infection, respectively (Fig. 3B), indi-
cating that the LD50of strain RB50?bscN is approximately
9.0 ? 106CFU, threefold greater than the LD50of strain RB50
(Fig. 3B and 1A). When mice were inoculated with 1 ? 105,
1.0 ? 106, or 2.0 ? 106CFU of 1289?bscN, 100%, 66%, and
0% of the mice survived the infection, respectively (Fig. 3B),
indicating that the LD50of strain 1289?bscN is approximately
1.2 ? 106CFU, 24-fold greater than that of strain 1289 (Fig. 3B
and 1B). Therefore, the TTSS appears to contribute more to
the virulence of strain 1289 than to that of strain RB50 (Fig.
1A, 1B, and 3B). Since the LD50of 1289?bscN is lower than
that of RB50?bscN, it suggests that another factor besides the
TTSS also contributes to the increased virulence of strain 1289
(Fig. 3B). Together, these data indicate that while the TTSS is
not the sole factor, it is partially responsible for the increased
virulence of strain 1289 compared to the virulence of strain
The TTSS is implicated in the increased virulence of ST32
strains. To examine whether isolates associated with B. bron-
chiseptica-related disease were of the same phylogenetic lin-
eage, we completed MLST and phylogenetic analyses using
two B. bronchiseptica strains from diseased hosts to determine
if they fell into the same ST as strain 1289. In addition to these
three disease-associated isolates, our analysis also included 58
additional Bordetella isolates, of which 55 were B. bronchisep-
tica strains (none known to be associated with diseased hosts
and all from a broad range of locations, dates, and hosts), 2 B.
pertussis strains, and 1 B. parapertussis strain (Fig. 4; see also
Table S1 in the supplemental material) (5, 11). These 58 iso-
lates served to demonstrate the genetic relatedness of strains
RB50 and 1289 and to confirm the evolutionary history of the
classical bordetellae. Consistent with other phylogenetic anal-
yses, both B. pertussis and B. parapertussis appear to have
evolved independently from B. bronchiseptica-like progenitors
(Fig. 4) (11, 42, 60). As previously described, strains RB50 and
1289 were identified as ST12 and ST32 isolates, respectively
(Fig. 4) (5, 11). Importantly, strains belonging to the same STs
as strains RB50 (11) and 1289 (Fig. 4; see also Table S1 in the
supplemental material) have been isolated from several conti-
nents, suggesting that these two STs exist worldwide. Two
isolates, strains S308 and S314, were selected for MLST anal-
ysis because they were collected from hosts with B. bronchisep-
tica-induced disease (see Table S1 in the supplemental mate-
rial). While one of these strains, S314, belongs to ST7, the
other strain, S308, belongs to the same ST as strain 1289 (Fig.
4). These data suggest that not all strains causing B. bronchi-
septica-induced disease are of the same phylogenetic lineage.
However, within the ST32 lineage, both strains with an accom-
panying pathology report (strains 1289 and S308) (Fig. 4) were
associated with B. bronchiseptica-induced disease, suggesting
that ST32 constitutes a more virulent lineage.
To determine if ST32 strains share increased virulence,
groups of three or four mice were inoculated with different
doses of strain S308 and were monitored for survival (see
criteria for strain selection in Materials and Methods) (Fig. 5).
While 0% of the mice survived an inoculation of 1 ? 105CFU,
100% survived an inoculation with 5 ? 104CFU, suggesting
that the LD50is approximately 7.5 ? 104CFU (Fig. 5), similar
FIG. 4. MLST analysis of 61 Bordetella strains. Unweighted pair
group method with arithmetic mean tree with 1,000 bootstraps
based on concatenated MLST gene sequences of 61 Bordetella iso-
lates (58 B. bronchiseptica, 2 B. pertussis, and 1 B. parapertussis
isolates). The identification number of each strain is listed. The
asterisks indicate strains that have undergone or are undergoing
full-genome sequencing (42). The ST is labeled next to each strain
and the complex is labeled next to each set of STs (complex I, which
includes B. bronchiseptica strains, complex II, which includes B.
pertussis strains, complex III, which includes B. parapertussis strains,
and complex IV, which includes B. bronchiseptica strains that ap-
pear to be most closely related to B. pertussis) as previously de-
scribed (5, 11). The numbers on the tree branches indicate branch
strength. All branch strengths below 50 were removed. The scale
indicates the relative genetic distances along the branches.
3974 BUBOLTZ ET AL.INFECT. IMMUN.
to that of strain 1289 (5 ? 104CFU) and approximately 40-fold
lower than that of strain RB50 (3 ? 106CFU) (Fig. 1A, 1B,
and 5). To determine if the TTSS contributed to the increased
virulence of strain S308, we deleted the bscN gene from this
strain and determined the LD50. When inoculated with 3 ? 106
or 1 ? 106CFU of S308?bscN, 0% and 100% of the mice,
respectively, survived the infection (Fig. 5). Therefore, the
LD50of this strain is approximately 2 ? 106CFU, which is
27-fold higher than that of its parental wild-type strain (Fig. 5).
These data indicate that the TTSS contributes more to the
virulence of strain S308 than to that of strain RB50 (Fig. 5 and
3B). Similar to strain 1289?bscN, the LD50of S308?bscN is
lower than that of RB50?bscN, which suggests that another
factor besides the TTSS also contributes to the increased vir-
ulence of strain S308. Together, these data suggest that the
increased virulence of ST32 strains is partially dependent on
MLST analysis has been completed for approximately 260
Bordetella strains, the vast majority of which are B. bronchisep-
tica (S.E. Hester, K. E. Creppage, M. C. Dunagin, K. Register,
and E. T. Harvill, unpublished data) (5, 11). Of these, only
three ST32 strains have been identified (Fig. 4), which suggests
that while these strains exist worldwide (see Table S1 in the
supplemental material), this ST may not contain as many
strains as other STs. Therefore, we wanted to determine if
other strains closely related to ST32 are more virulent than
strain RB50. We analyzed strain 448 from ST23, as it was the
strain most closely related to ST32 and having Bvg?morphol-
ogy that was available at the time of this study (Fig. 4). The
LD50of strain 448 was approximately 1 ? 106CFU, threefold
lower than that of strain RB50 (data not shown), and its bac-
terial loads in the lung were higher than those of strain RB50
(see Fig. S4 in the supplemental material) over the course of
the infection. Therefore, these data suggest that strains closely
related to ST32 are also more virulent than strain RB50 and
may represent lineages of increased virulence.
The severity of a B. bronchiseptica infection can range from
long-term asymptomatic carriage in the upper respiratory tract
to fatal pneumonia (18). While previous studies have corre-
lated differences in virulence or severity of disease to particular
bacterial factors (5, 39, 47), few studies have shown that these
factors actually contribute to the virulence of particular lin-
eages (4). Here, we identify a bacterial factor that contributes
to the increased virulence of a B. bronchiseptica lineage by
combining comparative genomic analyses, bacterial mutagen-
esis, phylogenetics, and a host infection model. B. bronchisep-
tica strain RB50, which was isolated from an asymptomatically
infected host, was less virulent than strain 1289, which was
isolated from a diseased host (Fig. 1; see also Table S1 in the
supplemental material) (8). Transcriptome analysis revealed
that TTSS-related genes were more highly expressed in strain
1289 than in strain RB50 (Fig. 2). Using allelic exchange, we
determined that the TTSS causes more-rapid cytotoxic effects
in macrophages, that there was negligible cytotoxicity in its
absence, and that it contributes more to the virulence of strain
1289 than to that of strain RB50 (Fig. 3). When assessing
another strain that belonged to the same ST as strain 1289 and
was also associated with B. bronchiseptica-induced disease, we
found that the increased virulence of this strain was also par-
tially attributable to the TTSS (Fig. 4 and 5). Combined, these
data suggest that the TTSS is involved in the increased viru-
lence of a B. bronchiseptica lineage.
The amount of genomic content shared between strains of a
single microbial species can vary substantially (30). The “core
genome” represents all genes shared between strains of the
same species, while the “flexible genome” includes those genes
that are variably present. The flexible genome is thought to
confer differences in phenotypes, such as virulence, host range,
and/or environmental niches, of different strains. Recently,
Cummings et al. proposed an analogous distinction to describe
those genes similarly expressed (the core regulon) or differen-
tially expressed (the flexible regulon) between strains, as not all
genes are similarly regulated by BvgAS among Bordetella
strains (10). The TTSS of B. pertussis, which mediates cellular
attachment rather than cytotoxicity, is expressed by some
strains but not others (14). Therefore, the TTSS of B. pertussis
appears to be part of the flexible regulon, as some B. pertussis
strains express the TTSS while others do not (14, 62). The work
described herein provides evidence that the TTSS is also part
of the flexible regulon of B. bronchiseptica, as strains of this
species express TTSS-related genes differentially. Together,
these data suggest that the TTSS can have different functions
and/or levels of these functions in different strains or species of
Since nearly all known TTSS-related genes were upregu-
lated in strain 1289 compared to their levels of expression in
strain RB50, we speculate that the underlying mechanism be-
hind the increased TTSS-mediated virulence of strain 1289 is
that increased TTSS gene expression leads to enhanced pro-
tein expression and secretion, which in turn increases TTSS-
mediated cytotoxicity and virulence. While TTSS-related genes
were upregulated in strain 1289, the genes encoding the master
regulator of the TTSS, bvgAS, were not differentially ex-
pressed. Since the TTSS is controlled by a complex, multilay-
ered, trans-regulatory gene network (34, 62), we propose that
the increased expression of a yet-unidentified, Bvg-activated
activator or decreased expression of a Bvg-activated repressor
may contribute to the differential expression of the TTSS be-
tween strains. This regulator may be one of the 23 upregulated
FIG. 5. TTSS-mediated effect on virulence of B. bronchiseptica
strain S308, a ST32 isolate. Groups of three or four C57BL/6 mice
were inoculated intranasally with the indicated doses of strain S308 or
S308?bscN. Survival curves were generated by inoculating mice with
the indicated dose and determining the percent survival over a 28-day
VOL. 77, 2009TTSS AND HYPERVIRULENCE OF A B. BRONCHISEPTICA LINEAGE 3975
or 51 downregulated transcriptional regulators identified in
Since 1289?bscN and S308?bscN are more virulent than
RB50?bscN, we conclude that the TTSS is not the only factor
that contributes to the increased virulence of ST32 strains.
Novel genes acquired via phage or horizontal gene transfer,
loss or downregulation of hypovirulence genes, or mutations in
strain 1289 may also contribute to this strain’s increased viru-
lence, although they do not appear to be sufficient for cytotox-
icity in vitro (15, 16). While many phage-related genes present
in strain RB50 were identified as absent in strain 1289, another
study of ours showed that a B. bronchiseptica strain lacking
these prophage genes is less virulent than strain RB50, making
it unlikely that the lack of these genes contributes to the in-
creased virulence of strain 1289 (5). A few known virulence-
related genes and many genes with unknown function were
identified as differentially expressed or divergent between
these strains. These genes may also contribute to the increase
in virulence of strain 1289 compared to that of strain RB50.
Differences in gene expression over the course of infection,
promoter mutations, or a gain of novel genes in strain 1289
would not be detected in our analyses and may also contribute
to the increased virulence of strain 1289. We are currently
sequencing the genome of strain 1289 and will then be able to
assess promoter mutations and novel genes that may play a
role in the increased virulence of this strain (A. M. Buboltz, X.
Zhang, S. C. Schu ¨ster, E. T. Harvill et al., unpublished data).
The most widely accepted view of virulence evolution as-
sumes that there is a cost-benefit trade-off to virulence, which
is defined as any reduction in host fitness following infection
(2, 12, 45). Under this framework, evolutionary processes that
lead to the maintenance of harmful effects are thought to be
characterized by the presence of other beneficial qualities (12).
Thus, a fitness-decreasing change in one trait in the pathogen
is accompanied by a fitness-increasing change in a different
trait (12). The ST32 strains described herein appear to be quite
successful, as they have been isolated from three separate
continents (South America, Europe, and the United States)
(see Table S1 in the supplemental material) (60). Here, we
show that ST32 strains appear to be associated with respiratory
disease and exhibit increased TTSS-mediated virulence, a po-
tential cost for the bacteria because pathogen success is de-
pendent upon host survival before transmission. Since the
TTSS increases colonization and persistence of B. bronchisep-
tica in the lungs of mice (44, 62), this effect may benefit the
bacteria in maximizing transmission, allowing for the selection
and maintenance of these highly virulent ST32 strains. Thus,
the fitness enhancement caused by increased TTSS-mediated
effects may be accompanied by the unavoidable side effect of
increased virulence (45).
A high degree of clonal diversity appears to exist among B.
bronchiseptica strains (3, 17, 19, 31, 36, 47). Recently, we re-
ported that strains belonging to ST27 and ST40, which were
collected from a wide geographical area, are hypovirulent and
have lost the genes encoding adenylate cyclase toxin, which was
previously believed to be among the few core factors required
for the success of the classical bordetellae (5). Here, we report
that the TTSS contributes to the increased virulence of strains
belonging to ST32, which appear to exist worldwide. Com-
bined, these studies support the conclusion that phylogenetic
lineages of B. bronchiseptica differentially regulate and utilize
distinct sets of virulence factors which can affect the overall
virulence of these STs. This versatility may contribute to the
wide variety in severity of respiratory disease observed upon B.
We thank Catherine Beckwith at the Pennsylvania State University,
College of Medicine; Bob Livingston at the University of Missouri
Research Animal Diagnostic Laboratory; David Relman at the De-
partment of Microbiology and Immunology, Stanford University; Frits
Mooi at the Laboratory for Vaccine-Preventable Diseases, National
Institute of Public Health and the Environment; and Gary Sanden at
the Center for Disease Prevention and Control for B. bronchiseptica
isolates. We thank all members of the Harvill laboratory for discussion
and critical review of the manuscript and Gra ´inne Long for assistance
with statistical analysis.
Mention of trade names or commercial products in this article is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the USDA.
This work was supported by NIH grants AI 053075, AI 065507, and
GM083113 (E.T.H.). The authors declare no conflicting financial in-
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