Streptococcus pneumoniae Serine Protease HtrA, but Not SFP or PrtA, Is a Major Virulence Factor in Pneumonia
Is a Major Virulence Factor in Pneumonia. PLoS ONE 8(11): e80062. doi:10.1371/journal.pone.0080062
Streptococcus pneumoniae Serine Protease HtrA, but Not SFP or PrtA, Is a Major Virulence Factor in Pneumonia
Sacha F. de Stoppelaar 0
Hester J. Bootsma 0
Aldert Zomer 0
Joris J. T. H. Roelofs 0
Peter W. M. Hermans 0
Cornelis van 't Veer 0
Tom van der Poll 0
Samithamby Jeyaseelan, Louisiana State University, United States of America
0 1 Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands, 2 Center for Experimental and Molecular Medicine (CEMM) , Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands , 3 Laboratory of Pediatric Infectious Diseases, Radboud University Medical Center , Nijmegen , The Netherlands, 4 Center for Molecular and Biomolecular Informatics , Radboud University Medical Center , Nijmegen , The Netherlands , 5 Department of Pathology, Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands , 6 Division of Infectious Diseases, Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands
Streptococcus (S.) pneumoniae is a common causative pathogen in pneumonia. Serine protease orthologs expressed by a variety of bacteria have been found of importance for virulence. Previous studies have identified two serine proteases in S. pneumoniae, HtrA (high-temperature requirement A) and PrtA (cell wall-associated serine protease A), that contributed to virulence in models of pneumonia and intraperitoneal infection respectively. We here sought to identify additional S. pneumoniae serine proteases and determine their role in virulence. The S. pneumoniae D39 genome contains five putative serine proteases, of which HtrA, Subtilase Family Protein (SFP) and PrtA were selected for insertional mutagenesis because they are predicted to be secreted and surface exposed. Mutant D39 strains lacking serine proteases were constructed by inframe insertion deletion mutagenesis. Pneumonia was induced by intranasal infection of mice with wild-type or mutant D39. After high dose infection, only D39DhtrA showed reduced virulence, as reflected by strongly reduced bacterial loads, diminished dissemination and decreased lung inflammation. D39DprtA induced significantly less lung inflammation together with smaller infiltrated lung surface, but without influencing bacterial loads. After low dose infection, D39DhtrA again showed strongly reduced bacterial loads; notably, pneumococcal burdens were also modestly lower in lungs after infection with D39Dsfp. These data confirm the important role for HtrA in S. pneumoniae virulence. PrtA contributes to lung damage in high dose pneumonia; it does not however contribute to bacterial outgrowth in pneumococcal pneumonia. SFP may facilitate S. pneumoniae growth after low dose infection.
Funding: This work was supported by an AMC PhD Scholarship to S.F. de Stoppelaar. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The bacterium Streptococcus (S.) pneumoniae is a major global cause
of human disease . S. pneumoniae is the most frequent cause of
community-acquired pneumonia and a common pathogen in
sepsis, the incidence being greatest at the extremes of age and in
immune compromised individuals . Although the discovery of
antibiotics and the development of vaccines have reduced the
health burden associated with pneumococcal infections, S.
pneumoniae still causes over 2 million deaths annually  and
increased bacterial resistance against currently available antibiotics
could make pneumococcal infections an even larger health threat
in the future . Consequently, additional knowledge about this
bacterium and its virulence factors is of importance.
In 1991, Courtney  was the first to describe a role for serine
proteases as virulence factors for S. pneumoniae. Since then, serine
protease orthologs have been found in many bacteria and
numerous roles in virulence and pathogenesis have been described
. One of these serine proteases is HtrA (high-temperature
requirement A), which has been identified as a virulence factor in
several bacterial species. In S. pneumoniae, HtrA is important for
bacterial stress response and protein quality control, and has a role
in competence [11,14,17]. As a posttranslational regulator, HtrA is
involved in bacteriocin activity and cell division [8,15,16]. HtrA
has been identified as an important virulence factor for S.
pneumoniae, i.e. HtrA deficient pneumococci demonstrated a
dramatically reduced virulence in models of pneumonia and
bacteremia . Another investigation identified PrtA (cell
wallassociated serine protease A) as a pneumococcal serine protease
important for virulence after intraperitoneal infection .
In this study, we searched for additional serine proteases as
virulence factors of S. pneumoniae. By reannotating the S. pneumoniae
D39 genome using a subsystems approach  and by screening
all proteins for the presence of serine protease associated domains
using Interproscan , we identified SFP (subtilase family
protein) as an additional surface-exposed pneumococcal serine
protease besides HrtA and PrtA, and generated directed gene
knockout mutants of htrA (SPD_2068), sfp (SPD_1753) and prtA
(SPD_0558) in S. pneumoniae strain D39. We tested their virulence
in an in vivo pneumonia model by inoculating mice with viable
wild-type (WT) and mutant S. pneumoniae via the airways, and
compared several outcome parameters 48h after infection.
Materials and Methods
Serine protease search
All proteins encoded in the genome of S. pneumoniae D39 where
screened for the presence of serine protease associated domains
using Interproscan . Domains IPR009003 and IPR001940
identified HtrA, domain IPR008357 identified SFP, while
domains IPR000209 and IPR015500 identified both SFP and
PrtA as predicted to encode serine proteinases. An additional
search for proteins predicted to have serine protease activity was
performed by examining membership of GO category 0008236,
resulting in the identification of SPD_1765 and SPD_1920. Blast
analysis of the proteome of S. pneumoniae D39 with HtrA, SFP and
PrtA with an E-value cut-off of 0.1 showed limited protein
sequence similarity of SFP with PrtA, but no other putative serine
proteases were identified. Furthermore the S. pneumoniae D39
genome was re-annotated using the +6RAST subsystems
approach  and the resulting annotations where searched for
protease encoding proteins. No other obvious serine proteases
could be detected using these methods. Subcellular localization
prediction of the putative serine proteases was performed with
Construction of directed deletion mutants
Directed-deletion mutants of S. pneumoniae D39 lacking HtrA
(D39DhtrA), SFP (D39Dsfp) or PrtA (D39DprtA) were generated by
allelic exchange of the target gene with a spectinomycin resistance
marker essentially as described previously . Briefly, an
extension PCR was performed to join 400500 bp 5 and 3
flanking sequences of the target gene with the spectinomycin
resistance cassette (obtained from pR412T7). The resulting PCR
products were introduced by competent stimulating peptide
(CSP1)-induced transformation into D39. Transformants were selected
on the basis of spectinomycin resistance and were checked by PCR
for recombination at the desired location on the chromosome.
Subsequently, the D39 WT strain was transformed with 1 mg
chromosomal DNA isolated from the mutants to prevent the
accumulation of inadvertent mutations elsewhere on the
chromosome. At the same time, D39 was mock-transformed to obtain a
coupled WT strain. All primers used in this study are shown in
In vitro growth assay
Mid-log growing mutant or WT S. pneumoniae were diluted to an
optical density (OD) of 0.1 (620 nm wavelength) in Todd Hewitt
broth (Oxoid microbiology products, Thermo Scientific,
Hampshire, UK) with 0.5% Yeast extract (THY). Cultures were
incubated at 37uC in a 5.0% CO2 incubator. OD was measured
every hour for the next 5 hours.
Specific pathogen-free C57BL/6 male and female mice were
purchased from Harlan Sprague-Dawley (Horst, the Netherlands).
Experimental groups were age- and sex matched, and housed in
the Animal Research Institute Amsterdam under standard care.
All experiments were conducted with mice between 10 and 12
weeks of age.
This study was carried out in concordance with the Wet op de
Dierproeven in the Netherlands. The Institutional Animal Care
and Use Committee of the Academic Medical Center approved all
experiments. All efforts were made to minimize suffering.
Induction of pneumonia happened under isoflurane anaesthesia.
Experimental study design
Pneumonia was induced by intranasal inoculation with S.
pneumoniae D39, D39DhtrA, D39Dsfp or D39DprtA (serotype 2;
56105 or 56104 colony forming units (CFU) in 50 mL isotonic
saline) using previously described methods . Mice were
euthanized 48 hours after induction of pneumonia (N = 8 mice
per group). Blood was obtained from the inferior vena cava and
diluted 4:1 with citrate. Bronchoalveolar lavage fluid (BALF), lung,
spleen and liver were harvested as described  and organs
were homogenised in five volumes of sterile isotonic saline. The
left lung lobe was fixed in 10% buffered formalin and embedded in
paraffin. Total cell numbers in BALF were determined by an
automated cell counter (Coulter Counter, Coulter Electronics,
Hialeah, FL, USA). Differential cell counts were performed on
cytospin preparations stained with a modified Giemsa stain
(DiffQuick; Dade Behring AG, Du dingen, Switzerland). For bacterial
quantification blood, BALF, and organ homogenates were serially
diluted by 10-fold in sterile isotonic saline and plated onto
sheepblood agar plates. Following 16 hours of incubation at 37uC CFU
were counted. For further measurements, homogenates were
diluted 1:1 with lysis buffer (300 mM NaCl, 30 mM Tris, 2 mM
MgCl2, 2 mM CaCl2, 1% (v/v) Triton X-100, pH 7.4) with
protease inhibitor mix and incubated for 30 minutes on ice,
followed by centrifugation at 680 g for 10 minutes. Supernatants
were stored at 20uC until analysis.
Four-micrometer sections of the left lung lobe were stained with
hematoxylin and eosin (H&E). Slides were coded and scored by a
pathologist blinded for group identity for the following parameters:
interstitial inflammation, endothelialitis, bronchitis, oedema,
pleuritis and presence of thrombi. All parameters were rated
separately from 0 (condition absent) to 4 (most severe condition).
The total histopathological score was expressed as the sum of the
scores of the individual parameters, with a maximum of 24. In
addition, pulmonary infiltrate was scored as a percentage of total
lung surface occupied by confluent infiltrates.
Interleukin (IL)-6, tumor necrosis factor alpha (TNF-a),
keratinocyte-derived cytokine (KC) and IL-1b were measured
using commercially available ELISA kits (R&D Systems,
Abingdon, UK). Myeloperoxidase (MPO; Hycult, Uden, the
Netherlands) was measured by ELISA according to manufacturers
Data are expressed as box and whisker plots showing the
smallest observation, lower quartile, median, upper quartile and
largest observation, or as medians with interquartile ranges.
Comparisons between groups were first performed using
KruskalWallis one-way analysis of variance test; in case of significant
differences, differences between groups were tested using the
Mann-Whitney U test. All analyses were done using GraphPad
Prism version 5.01 (GraphPad Software, San Diego, CA). P-values
, 0.05 were considered statistically significant.
Primers for generation of directed mutants
SpecR cassette control primer
htrA left flanking region
htrA left flanking region
htrA right flanking region
htrA right flanking region
htrA gene control primer
sfp left flanking region
sfp left flanking region
sfp right flanking region
sfp right flanking region
sfp gene control primer
prtA left flanking region
prtA left flanking region
prtA right flanking region
prtA right flanking region
prtA gene control primer
S. pneumoniae D39 genome contains three putative
To identify all serine protease proteins encoded in the genome
of S. pneumoniae D39, we screened the D39 proteome for the
presence of serine protease domains using Interproscan as
described in the Experimental procedures. Furthermore, the
D39 genome was re-annotated to identify additional proteases.
This led to the identification of several proteases, of which only
HtrA (SPD_2068), PrtA (SPD_558) and SFP (SPD_1753) were
predicted to have both serine protease activity (figure 1A-C) and
be secreted and surface-exposed based on the presence of a signal
sequence . The predicted PDZ (Post synaptic density protein,
Drosophila disc large tumor suppressor, and Zonula occludens-1
protein) domain in HtrA could be involved in protein-protein
interactions. The DUF (Domain of Unknown Function) 1034
domain in PrtA is predicted to have serine-type endopeptidase
activity. Two additional putative serine proteases were identified
by their membership of GO category 0008236 (SPD_1765 and
SPD_1920), but they were excluded from further analysis because
they did not contain a signal sequence or a cell surface exposed
serine protease domain. BLAST analysis of the D39 proteome
with HtrA, PrtA and SFP did not reveal any additional putative
serine protease encoding genes.
S. pneumoniae D39DhtrA, but not D39Dsfp or D39DprtA,
displays diminished growth and dissemination in vivo
Mutant S. pneumoniae strains did not show reduced growth
compared to WT S. pneumoniae in THY at 37 uC and 5.0% CO2
(figure 1D). To study the role of the three serine proteases in S.
pneumoniae virulence, we infected mice with 56105 CFU of either
WT D39, D39DhtrA, D39Dsfp or D39DprtA via the airways and
CCACTAGTTCTAGAGCGGCAAACTACCCAAGGCTCCAC GACTTGCCCATCTTATCTGC GCGTCAATTCGAGGGGTATCACCATTCTATCGGAGACACC TCGCTGAGAATCTGTGTCAG
quantified bacterial loads at the primary site of infection (lungs)
and distant body sites (blood, spleen and liver) 48 hours later
(figure 2). WT S. pneumoniae D39, D39Dsfp and D39DprtA had
multiplied in the lungs, accompanied by dissemination to spleen
and liver; the counts of these three pneumococcal strains were
similar in all body sites examined. In contrast, D39DhtrA counts
were markedly lower in the lungs and in all distant body sites when
compared with the other three strains (P , 0.0005). In addition,
no bacteria could be detected in the blood of D39DhtrA infected
animals (P , 0.005).
Reduced lung inflammation during pneumonia caused
by S. pneumoniae D39DhtrA or D39DprtA
This model of pneumococcal pneumonia is associated with
histological features in the lung characteristic for lower respiratory
tract infection, including interstitial inflammation, endothelialitis,
edema, inflammatory infiltrates and pleuritis . To determine
the impact of the three S. pneumoniae serine proteases on the
induction of these inflammatory alterations, we semi-quantitatively
scored lung histology slides after pneumonia caused by WT D39,
D39DhtrA, D39Dsfp or D39DprtA (figure 3). The lungs of D39DhtrA
infected mice displayed significantly less inflammation and a
smaller infiltrated lung surface (P , 0.01). Remarkably, in spite of
similar bacterial loads, the lungs of D39DprtA infected mice also
displayed lower histopathology scores when compared with lungs
of WT D39 infected mice, together with a smaller infiltrated lung
surface (P , 0.05). Considering that neutrophils play a key role in
the inflammatory response during respiratory tract infection by S.
pneumoniae [1,26], we next determined total cell and neutrophil
counts in BALF after infection with WT D39, D39DhtrA, D39Dsfp
or D39DprtA (figure 4). We additionally measured MPO
concentration in whole lung homogenates, as a marker of neutrophil
content of lung tissue. Total cell counts were lower in BALF
obtained from D39DhtrA infected mice (P , 0.05); a similar trend
was observed for neutrophil influx. In accordance, whole lung
MPO concentrations were lower after infection with D39DhtrA (P
, 0.05). The deletion of either sfp or prtA did not influence cell
recruitment. Cytokines and chemokines play an eminent role in
the regulation of inflammation during pneumonia [1,26].
Therefore, we measured cytokines (TNFa, IL-1b, IL-6) and a chemokine
(KC) in whole lung homogenates as an additional readout for
pulmonary inflammation. Lung IL-1b, IL-6 and KC levels were
lower in D39DhtrA when compared with the other three S.
pneumoniae strains (table 2).
Modest role for SFP after low dose infection
The data presented above confirm that HtrA plays a significant
role in S. pneumoniae virulence . We argued that the
Figure 2. S. pneumoniae D39DhtrA, but not D39Dsfp or D39DprtA, displays diminished growth and dissemination in vivo. Mice were
infected with WT or mutant S. pneumoniae (56105 CFU) via the intranasal route and euthanized 48 hours later. Bacterial counts were determined in
lung (A), blood (B), spleen (C) and liver (D). Data are expressed as box- and whisker plots depicting the smallest observation, lower quartile, median,
upper quartile and largest observation. N = 8 mice per group at each time point. *** P , 0.005 versus WT S. pneumoniae.
contribution of PrtA and SFP to S. pneumoniae virulence could be
more subtle and as such not noticed in our model with a relatively
high bacterial dose. Accordingly, we repeated our infection
experiments with a 10-fold lower inoculum (56104 CFU). These
studies again revealed the reduced virulence of D39DhtrA, as
reflected by lower bacterial loads in lungs (P , 0.005) and liver (P
, 0.005) at 48 hours post infection (figure 5). Of interest,
pneumococcal burdens were also modestly but significantly lower
in lungs after infection with D39Dsfp (P , 0.05); this difference
Lung homogenate, t = 48h (pg/mL)
Mice were infected with the S. pneumoniae strain indicated (56105 CFU) via the intranasal route and euthanized 48 hours later. Data are expressed as medians and
interquartile range of 7 or 8 mice per group. ** P , 0.01 versus WT S. pneumoniae.
with WT D39 was not observed in blood or distant organs. Cell
influx in low dose pneumonia was modest (Figure 6), with less than
half of the total cell counts in BALF compared to high dose
infection. No difference in total cell numbers was observed
between WT or mutant S. pneumoniae infected mice (figure 6A).
Neutrophil influx determined by cell differentiation on cytospin
slides however was diminished in D39DhtrA infected animals
(figure 6B) (P , 0.05).
Serine protease orthologs have been found in many bacteria,
contributing to their virulence to a significant extent [4-14].
Previous research identified the serine protease HtrA as a major
virulence factor in S. pneumoniae during experimentally induced
pneumonia [11,14]. We here sought for additional S. pneumoniae
serine proteases and determined their role in virulence during
respiratory tract infection in vivo. Our main findings are that the S.
pneumoniae D39 genome expresses three putative secreted,
surfaceexposed serine proteases: HtrA, SFP and PrtA. We confirmed
reduced virulence after high and low dose infection of D39DhtrA
[11,14], as reflected by strongly reduced bacterial loads,
diminished systemic dissemination and decreased lung inflammation.
After high dose infection D39DprtA induced significantly less lung
inflammation without influencing bacterial loads. Pneumococcal
burdens were also modestly but significantly lower in lungs after
low dose infection with D39Dsfp. These data reveal two additional
pneumococcal serine proteases that can modify the host response
during pneumonia, albeit clearly to a more modest extent than
Structure and function of HtrA have been widely studied. S.
pneumoniae HtrA has been identified to help bacteria survive
environmental pressures such as elevated temperature, oxidative
stress, and osmotic stress . Another study revealed an
important role for HtrA during S. pneumoniae cell division .
HtrA additionally has a distinct role in bacteriocin activity by
reducing pneumocin expression [8,16]. Pneumocins mediate
intraand interspecies competition in vitro and have been shown to
provide a competitive advantage in vivo . In the present study,
we confirmed the strongly reduced virulence of D39DhtrA in
pneumonia . D39DhtrA infected animals displayed more than
3 logs lower bacterial counts in their lungs at 48 hours after
infection; in addition, their lungs showed minimal signs of lower
respiratory tract infection upon histopathological examination.
There was significantly less dissemination to distant organs in
D39DhtrA infected mice. Notably, D39DhtrA could be detected in
spleen and liver, although blood cultures were sterile in all
experiments. Ibrahim et al  concluded that D39DhtrA S.
pneumoniae did not disseminate from the lungs based on negative
blood cultures; our data, however, indicate that D39DhtrA is able
to spread to distant body sites.
It is known that apolactoferrin can kill many species of bacteria,
including Streptococcus pneumoniae. Lactoferricin, an N-terminal
peptide of apolactoferrin, and fragments of it are even more
bactericidal than apolactoferrin. PrtA cleaves apolactoferrin,
greatly enhancing the killing activity of apolactoferrin and its
cleavage products . Thus, in theory, PrtA deficiency might
cause increased rather than decreased virulence due to a
diminished capacity of apolactoferrin to kill S. pneumoniae.
Nonetheless, the only in vivo study performed thus far with PrtA
deficient S. pneumoniae showed decreased virulence compared to
WT S. pneumoniae when injected intraperitoneally in mice . Our
pneumonia model is more relevant to determine the role of PrtA in
S. pneumoniae virulence, as pneumonia is the primary illness caused
by pneumococci. In both high and low dose infection, bacterial
outgrowth, pulmonary cell influx and cytokine release were similar
after induction of pneumonia with D39DprtA or WT S. pneumoniae.
Interestingly, histopathology scores of lungs and the percentage of
infiltrated lung surface were significantly lower in D39DprtA
infected animals than in mice inoculated with WT S. pneumoniae,
suggesting that PrtA has a modest role in the induction of
pulmonary inflammation during pneumococcal pneumonia
without influencing cellular influx or cytokine release in the lungs, and
bacterial multiplication and dissemination.
To our knowledge, the role of SFP in pneumococcal virulence
has not been studied before. SFP has homology with S. agalactiae
CspA, a serine protease capable of inactivating chemokines in vitro
. While after high dose infection the growth of D39Dsfp was
Figure 4. Diminished cell and neutrophil influx during pneumonia caused by S. pneumoniae D39DhtrA. Mice were infected with WT or
mutant S. pneumoniae (56105 CFU) via the intranasal route and euthanized 48 hours later. Cell (A) and neutrophil (B) influx were determined on BALF
cytospin preparations. As a marker of neutrophil influx in lung tissue, MPO was measured in whole lung homogenates (C). Data are expressed as
boxand whisker plots depicting the smallest observation, lower quartile, median, upper quartile and largest observation. N = 8 mice per group at each
time point. * P , 0.05 versus WT S. pneumoniae.
indistinguishable from that of WT S. pneumoniae, this mutant strain
demonstrated slightly reduced growth in the lungs in low dose
pneumonia. This modest role of SFP mediated virulence should be
confirmed in additional studies using different S. pneumoniae strains.
We identified two additional hypothetical proteins, SPD_1765
and SPD_1920, predicted to have serine protease activity. These
were not examined further here as their serine protease domain
was likely not exposed to the outer cell surface. It may be of
interest to examine the roles of intracellular serine protease activity
for SPD_1765 and SPD_1920 in S. pneumoniae virulence in future
The current investigation addressed the role of three serine
proteases expressed by S. pneumoniae (HtrA, SFP and PrtA) in
virulence in vivo by comparing bacterial growth and dissemination
and the accompanying inflammatory response in the lung after
48 hours of airway infection by newly generated deletion mutants.
This time point was selected since our main endpoint of interest
was bacterial growth; the 48-hour time point reflects late stage
pneumonia shortly before mice are expected to die. Our data
confirm the previously reported important role for HtrA in S.
pneumoniae virulence . In this previous paper, reconstitution of
HtrA into D39DhtrA reverted D39DhtrA to its full virulence .
We here created a new HtrA deletion mutant and our
investigation is limited by the fact that we did not reconstitute
HtrA in our independently generated D39DhtrA strain. Of note,
however, our main objective was to identify new pneumococcal
serine proteases as virulence factors in pneumonia. Since our
results only reveal a significant role for the already established
HrtA , we think that the absence of a HrtA reconstitution
experiment does not jeopardize our main conclusion (i.e., that the
other proteases identified do not significantly contribute to the
virulence of S. pneumoniae). In contrast to its reported role in
intraperitoneal infection  we here show that PrtA does not
influence bacterial multiplication and dissemination in
pneumococcal pneumonia, however, PrtA had a modest role in the
induction of pulmonary inflammation. Finally, in the first studies
reported to date, we provide evidence that SFP may facilitate S.
pneumoniae growth after low dose infection of the lower respiratory
tract, although clearly these data need to be confirmed in
independent experiments involving more time points. Together
these data firmly establish that virulence of S. pneumoniae is
dominated by HtrA.
Figure 5. HtrA strongly contributes to virulence after low dose infection with a more modest role for SFP. Mice were infected with a
10fold lower inoculum of WT or mutant S. pneumoniae (relative to the infectious dose used in the experiments shown in figures 24; 56104 CFU) via the
intranasal route and euthanized 48 hours later. Bacterial counts were determined in lung (A), blood (B), spleen (C) and liver (D). Data are expressed as
box- and whisker plots depicting the smallest observation, lower quartile, median, upper quartile and largest observation. N = 8 mice per group at
each time point. * P ,0.05, ** P , 0.01, *** P , 0.005 versus WT S. pneumoniae.
Figure 6. Diminished cell and neutrophil influx during pneumonia caused by low dose S. pneumoniae D39DhtrA. Mice were infected
with a 10-fold lower inoculum of WT or mutant S. pneumoniae (relative to the infectious dose used in the experiments shown in figures 24; 56104
CFU) via the intranasal route and euthanized 48 hours later. Cell (A) and neutrophil (B) influx were determined on BALF cytospin preparations. Data
are expressed as box- and whisker plots depicting the smallest observation, lower quartile, median, upper quartile and largest observation. N = 8
mice per group at each time point. * P , 0.05 versus WT S. pneumoniae.
Conceived and designed the experiments: SFdS PWMH CvtV TvdP.
Performed the experiments: SFdS HB. Analyzed the data: AZ JJTHR.
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