Pilus 2

Download as pdf or txt
Download as pdf or txt
You are on page 1of 17

RESEARCH ARTICLE

A Serine-Threonine Kinase (StkP) Regulates


Expression of the Pneumococcal Pilus and
Modulates Bacterial Adherence to Human
Epithelial and Endothelial Cells In Vitro
Jenny A. Herbert, Andrea M. Mitchell, Timothy J. Mitchell*
Institute of Microbiology and Infection, School of Immunity and Infection, University of Birmingham,
Birmingham, England, United Kingdom

* t.j.mitchell@bham.ac.uk

Abstract
The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the
pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of in-
OPEN ACCESS fection. The gene for this regulator is located adjacent to the gene for its cognate phospha-
tase in the pneumococcal genome. The phosphatase dephosphorylates proteins
Citation: Herbert JA, Mitchell AM, Mitchell TJ (2015)
A Serine-Threonine Kinase (StkP) Regulates phosphorylated by StkP and has been shown to regulate a number of key pneumococcal
Expression of the Pneumococcal Pilus and virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adher-
Modulates Bacterial Adherence to Human Epithelial ence of pneumococci to human cells has not previously been reported. In this study we
and Endothelial Cells In Vitro. PLoS ONE 10(6):
e0127212. doi:10.1371/journal.pone.0127212
show StkP represses the pneumococcal pilus, a virulence factor known to be important for
bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adher-
Academic Editor: Eliane Namie Miyaji, Instituto
Butantan, BRAZIL
ence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial
cells. This suggests that the pneumococcal pilus may play a role in adherence during infec-
Received: February 2, 2015
tions such as meningitis and pneumonia. We show that regulation of the pilus occurs at the
Accepted: April 2, 2015 population level as StkP alters the number of pili-positive cells within a single culture. As far
Published: June 19, 2015 as we are aware this is the first gene identified outside of the pilus islet that regulates the bi-
Copyright: © 2015 Herbert et al. This is an open phasic expression of the pilus. These findings suggest StkPs role in cell division may be
access article distributed under the terms of the linked to regulation of expression of a cell surface adhesin.
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.

Data Availability Statement: All primary data are


Introduction
available in the manuscript and supplementary
information files. Streptococcus pneumoniae is normally found as a commensal in the nasopharynx of humans,
Funding: This work was funded by a scholarship
colonising 10% to 40% of adults and children respectively [1]. This bacterium can also cause a
from the BBSRC to JAH. The funders had no role in number of invasive diseases including meningitis, septicaemia, pneumonia and otitis media.
study design, data collection and analysis, decision to The organism is the worlds biggest killer of children under the age of five, and is a huge burden
publish, or preparation of the manuscript. on the health services worldwide [2]. Current vaccines target the pneumococcal capsule. How-
Competing Interests: The authors have declared ever there are over 90 different capsule types and only a small proportion of these can be in-
that no competing interests exist. cluded in the vaccine composition [3]. Vaccine replacement and capsule switching adds to the

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 1 / 17


Regulation of the Pneumococcal Pilus

limitations of this type of vaccine in the longer term [4–8]. The high cost of this vaccine also
prevents its usage in countries with the highest burden of disease.
The ability of the pneumococcus to cause invasive disease is attributed to its large cache of
virulence factors (see review [9]). This includes genes encoding two types of pilus (type 1 and
type 2). The type 1 pneumococcal pilus is encoded on a 12kb genomic islet (PI-1) and is pres-
ent in approximately 30% of pneumococcal strains [10]. The type 2 pilus is encoded on a 6.6kb
island (PI-2) encoding 5 genes and is present in approximately 16% of strains [11]. Pilus 2 ex-
pression has a marginal effect on bacterial adhesion to epithelial cells [11]. The PI-1 islet con-
tains 7 genes, three encoding pilus structural proteins (RrgA, RrgB and RrgC), three encoding
the sortase enzymes that are required for pilus assembly (SrtB, SrtC and SrtD) and one encod-
ing a positive regulator of the islet (RlrA) [12,13]. There are 4 promoters; one upstream of rlrA,
rrgA, rrgB and srtB which may allow differential expression of these genes [14]. The type 1
pilus has been shown to play a role in host cell adhesion, biofilm formation, and pathogenesis
of pneumonia and is being studied as a potential vaccine candidate [12,15–18]. Recent findings
have also implicated the type 1 pilus in modulating host immune defence by interacting with
TLR2 and complement receptor 3 [19,20]. Regulation of the type 1 pneumococcal pilus is com-
plex potentially involving two transcriptional regulators and six two-component signal trans-
duction systems (TCS) [21–25]. The pilus also shows a biphasic pattern of expression with
roughly 30% of bacteria in a single population expressing the pilus on the cell surface at any
one time. The proportion of cells expressing the pilus is strain dependant. The regulatory
mechanism controlling this biphasic pilus expression is complex and is mediated by expression
of rlrA and a positive feedback loop of the regulator RlrA [26,27].
The pneumococcus has a single eukaryotic like serine/threonine protein kinase which is a
global regulator that regulates genes involved in natural competence development, cell wall
biosynthesis, oxidative stress response and metabolism [28,29]. The importance of this pro-
tein has been shown by gene deletion experiments. A stkP deletion mutant has decreased
ability to survive under stress induced conditions (heat/ osmotic/ oxidative and acid stress),
reduced transformation efficiency and decreased virulence in vivo in a bacteraemia and
pneumonia model of infection [29,30].
StkP contains an N-terminal kinase domain joined by a hydrophobic linker to an extracellu-
lar domain [28,31] that consists of four penicillin binding protein and serine threonine kinase
associated (PASTA) domains [32]. PASTA domains in penicillin binding protein 2x (PBP2x)
are thought to recognise the amount of unlinked peptidoglycan and regulate the amount of
cross-linking via its transpeptidase domain [33,34]. These domains are the target of β-lactam
antibiotics blocking cell wall biosynthesis leading to bacterial cell death [35,36]. The PASTA
domains in StkP have also been shown to bind to synthetic peptidoglycan and β-lactam antibi-
otics [32,37]. This binding has been shown to directly affect the activity of the kinase domain,
and the level of phosphorylation of target proteins. Furthermore, the PASTA domains have
been shown to be important in localisation of StkP to the cell septum [37,38].
StkP modulates expression via phosphorylation of target proteins on a serine or threonine
residue [39]. Current known targets of StkP include a number of cell division proteins and it
has also been shown to phosphorylate response regulator CbpS (RR06) and response regulator
RitR (ORR) [40–42].
Here we show that StkP represses expression of the type 1 pneumococcal pilus in a sero-
type 4 strain. This regulation modulates adherence to human brain microvascular endothelial
cells and human lung epithelial cells. Complementation of an stkP knockout mutant with the
full length stkP gene reverted the level of adherence to wild-type. Expression of a variant of
the StkP protein lacking a single PASTA domain caused partial reversion of adherence

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 2 / 17


Regulation of the Pneumococcal Pilus

demonstrating the importance of the PASTA domain to StkP signalling. This regulation of
the pilus was shown to occur at the population level.

Materials and Methods


Bacterial strains and growth conditions
The bacterial strains used in this study are described in S1 Table. S.pneumoniae strains were
cultured on blood agar base (BAB, Oxoid) with 5% Horse blood (E&O laboratories) at 37°C
with 5% CO2. Liquid cultures were prepared by growth at 37°C in brain heart infusion broth
(Oxoid) to OD600nm 0.6. Media were supplemented with kanamycin (400μg/ml), spectinomy-
cin (200μg/ml) or chloramphenicol (10μg/ml) depending on the selectable marker present in
the strain. E.coli strains were grown on Luria Bertani (LB) agar or in LB broth containing the
appropriate antibiotic, ampicillin (100μg/ml) or kanamycin (100μg/ml).

In vitro mariner mutagenesis


MarC9 transposase was purified as previously described [43,44]. The reaction mixture con-
sisted of 1μg of stkP PCR product, 10μl 2x transposition buffer (10% glycerol, 2mM DTT,
25mM Hepes pH 7.9, 250μg/ml BSA, 100mM NaCl, 10mM MgCl2), 1μg pR412 plasmid DNA,
0.5μl transposase enzyme and made to 20μl with PCR grade water. The reaction mix was incu-
bated for 6 hours at 30°C. The reaction was cleaned using the Wizard SV gel and PCR clean up
system (Promega) as per manufacturers guide and heated for 10 minutes at 75°C to inactivate
the transposase. To the eluate 10μl 10x T4 DNA reaction buffer, 1.5μl 2mM dNTPS, 2μl T4
DNA polymerase (3U/μl) (NEB) and 0.5μl 10mg/ml BSA were added and incubated at 16°C
for 30 minutes followed by 75°C for 10 minutes to inactivate the enzyme. Finally 2μl of 1mM
NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E.coli DNA ligase (NEB) was
added, incubated at 16°C overnight and then stored at 4°C until transformation. The whole re-
action mix was subsequently used to transform an unencapsulated TIGR4 strain.

Construction and verification of gene knockouts and complemented


strains
All primers used for construction of mutants and complemented strains are listed in S2
Table. Construction of T4ΔstkP was performed via in vitro mariner mutagenesis. The full
stkP gene was amplified from TIGR4 using primers stkPF FL and stkPR. In vitro mariner mu-
tagenesis reactions were performed as described above. The initial mutant was constructed in
an unencapsulated TIGR4 strain due to its increased transformation efficiency [45]. The re-
sulting mutant contained a transposon insertion in stkP containing a spectinomycin resis-
tance cassette. The position and directionality of the insert was confirmed by using primers
MP127 and MP128. To transfer the insertion into TIGR4 primers stkPF FL and stkPR were
used to amplify stkP containing the transposon insertion, which was subsequently trans-
formed into TIGR4 creating T4ΔstkP.

Splice overlap PCR


Construction of an RrgB deletion mutant was performed via splice overlap PCR replacing the
whole rrgB gene with a kanamycin resistance cassette amplified from pR410 plasmid [46].
Three primer pairs were required amplifying the upstream (0462F-25C/ 0462 R) and down-
stream (0464F/ 0464 R-34R) of rrgB and the kanamycin resistance cassette from pR410
(KANGB-F/ KANGB-R). Fragments were transformed into TIGR4 and selected on BAB

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 3 / 17


Regulation of the Pneumococcal Pilus

containing kanamycin. Gene deletion was confirmed by sequencing. For construction of


T4ΔstkPΔrrgB the ΔrrgB fragment was amplified from T4ΔrrgB and transformed into T4ΔstkP.

Construction of StkP complemented strains


StkP complemented strains were constructed placing either the full length StkP amplified from
TIGR4 or 3rd PASTA domain deletion StkP (amplified from strain Xen35) downstream of a
strong promoter chromosomally inserted into SP_1886 in T4ΔstkP. A modified version of
pCEP2 [47] was used in which a strong promoter identified from RNA-seq analysis of TIGR4
was inserted. To create T4ΔstkPrST and T4ΔstkPrXST plasmids pCP2 ST and pCP2 XST
were transformed into T4ΔstkP respectively. Integration into the genome at the position of
SP_1886 was confirmed by PCR using primers flanking the insertion site (S2 Table).

Pneumococcal transformation
Pneumococcal transformation was performed as described in [48], except CSP-2 was used and
BAB plates supplemented with the desired antibiotic, for T4ΔstkP spectinomycin (200μg/ml),
T4ΔrrgB kanamycin (400μg/ml) and T4ΔstkPrST/ T4ΔstkPrXST chloramphenicol (10μg/
ml). For plasmid and PCR product transformation 1μg of DNA was used. For in vitro mariner
mutagenesis the whole final reaction was used for transformation.

Isolation of pneumococcal RNA


For RNA extraction pneumococcal cultures were grown in triplicate in BHI to OD600nm 0.6.
RNA extraction was performed as described in [22], with the following exceptions. After the
initial lysis steps extraction was performed using the RNeasy mini kit (Qiagen), as per the man-
ufacturers instructions. Post extraction a further DNase step was performed using the TURBO
DNA-free kit (Ambion, Life Technologies, UK) as per the manufacturers guide. RNA was
stored at -80°C until required.
For cDNA synthesis 2μg of total RNA was used and 1μl of random primers added (Life
Technologies) and made to 11μl with nuclease free water. Samples were incubated at 70°C for
10 minutes and then snap cooled on ice. To each sample 5μl 5x first strand buffer, 2.5μl DTT
(100mM), 2.3μl dNTP mix (5mM dGTP/ dATP/ dTTP/ dCTP); 1.7μl nuclease free water and
2.5μl Superscript 11 (200U/μl) were added. Samples were incubated at 25°C for 10 minutes fol-
lowed by 45°C for 90 minutes.

RT-PCR
All primers used for RT-PCR analysis are described in S3 Table. cDNA synthesis was per-
formed as described above.
Real-time PCR was performed using FastStart Universal SYBR green master mix (ROX)
(Roche) as per manusfacturers instructions. All samples were analysed on a Chromo4 system
CFB-3240 (Bio-Rad, USA). Reaction conditions consisted of an initial incubation of 2 minutes
at 50°C, followed by 10 minutes at 95°C. 40 cycles of 95°C for 15 seconds, 55°C for 30 seconds
and 72°C for 30 seconds were performed followed by a final 5 minutes at 72°C. gyrA was used
as an internal control to normalise for cDNA synthesis variations.
Analysis was performed in Opticom Monitor version 3.1. Background was subtracted in the
software from No-RT controls and replicates grouped together with at least two replicates used
for analysis. Data was analysed using the 2-ΔΔCT method [49]. Graphical data representation
was performed in Prism version 4.0b (GraphPad Software), each bar representing the sample
replica means ± standard deviation error bars.

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 4 / 17


Regulation of the Pneumococcal Pilus

Western blotting
For all western blots S.pneumoniae strains were grown to OD600nm 0.6 in BHI. The bacterial ex-
tracts were run on NuPAGE1 Novex 4–12% Bis-Tris gels and proteins were transferred using
the iBlot1 module. The membrane was blocked and then incubated with a 1/4000 dilution of
the primary antibody. Primary antibody either consisted of an in house Mouse Anti- RrgB,
Rabbit Anti- RrgA, Rabbit Anti- StkP, Rabbit Anti- PhpP and Rabbit Anti- GroEL (E.coli) pAb
(Enzo Life Sciences, UK). Primary antibody was detected using a 1/20,000 dilution of the HRP
labelled secondary antibody Goat Anti-Rabbit IgG HRP linked F(ab)2 (GE Healthcare) or Goat
Anti-Mouse IgG HRP (Southern Biotech) and developed using Immobilon Western Chemilu-
minescent HRP substrate (Millipore).

Flow cytometry
S.pneumoniae strains were fixed in 1ml 2% paraformaldehyde (Sigma-Aldrich, UK). Antibody
staining was performed as described in [27]. Double antibody staining was performed using
Mouse Anti- RrgB polyclonal (raised in house) and Rabbit Anti- Type 4 capsule (Statens
Serum Institute) followed by secondary antibody staining with Goat Anti-Rabbit IgG (H&L
chain specific) Allophycocyanin (APC) conjugate (Southern Biotech) or Goat Anti-Mouse IgG
(γ chain specific) Fluorescein (FITC) Conjugate (Southern Biotech).
Labelled samples were analysed on a FACScalibur flow cytometer using CellQuest-Pro soft-
ware (BD biosciences). FACS analysis was performed in FlowJo 9.4.10 for Macintosh (Tree Star).

Adherence assay
HBMEC (Human brain microvascular endothelia cells) were grown in Advanced RPMI 1640
media (Life technologies) supplemented with 20% FBS (Fetal bovine serum (EU approved),
Biosera), 2mM L-Glutamine (Sigma Aldrich), 1% 100X Penicillin streptomycin solution
(Sigma Aldrich,), and 1% Fungizone Antimycotic (Gibco). A549 (Human lung epithelial carci-
noma cell line, ATCC-CCL-185) cells were grown in Hams F12K (Kaighns modification)
media (Life Technologies) supplemented with 10% FBS, Penicillin streptomycin and Fungi-
zone as above.
For adherence assay all cell lines were grown in the same media as stated above however the
penicillin streptomycin solution and Fungizone1 was omitted. All adherence assays were per-
formed in 24 well microtitre plates. 2x105 viable cells were seeded into each well and made to
1ml with media. The plates were incubated at 37°C in 5% CO2 for 48 hours (cells confluent).
107 CFU of S.pneumoniae taken directly from a frozen bacterial stock were seeded in 1ml tissue
culture media into a single well containing tissue culture cells. The assay was incubated at 37°C
in 5% CO2 for 2 hours when viable counts were performed giving the number of non-adherent
bacteria. Cells were then washed 3 times and solublised in 0.0125% Triton-X-100 (Sigma-Al-
drich) and the percentage of adherent bacteria calculated. Graphical presentation and statistical
analysis was performed in Prism version 4.0b (GraphPad Software). Statistical analysis was
performed using a one-way ANOVA with Tukey’s multiple comparison test.

Results
A gene deletion of stkP (T4ΔstkP) was constructed in a serotype 4 strain (TIGR4). Comple-
mented strains were constructed expressing full length StkP (TIGR4) or the 3rd PASTA domain
deleted allelic variant of StkP (referred to as XST) [47,50,51]. The full length and allelic variant
amino acid sequence of StkP are shown in S1 Fig. Deletion of the whole 3rd PASTA domain in
this strain has occurred between two almost identical 10 amino acid repeats located at the end

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 5 / 17


Regulation of the Pneumococcal Pilus

of the second and third PASTA domain. The kinase is highly conserved within the pneumococ-
cus and shows 99.5% amino acid sequence homology between strains [52]. Use of this natural
variant avoided the need for complex cloning. There is currently no crystal structure available
to evaluate what the effect of this deletion may have on StkP structure. However schematic
models imply the PASTA domains likely span the width of the peptidoglycan cell wall [38]. Re-
moval of one of these domains would likely shorten the length the domains span beyond the
cell wall and may affect interaction between the PASTA domains and their extracellular stimu-
lus, in turn altering kinase activation.

Regulation of the pilus by StkP


Initial experiments were performed evaluating expression changes in the 7 pilus islet genes in
the single StkP knockout in TIGR4 and the StkP complemented strains (Fig 1) [13,17].
In the StkP knockout RT-PCR analysis showed an up-regulation of all genes present on the
pilus islet compared to TIGR4, suggesting that StkP normally functions to repress expression
of the pilus islet genes. When the full length StkP was expressed in T4ΔstkP (T4ΔstkPrST) this
reverted the effects and there was a decrease in the expression of the pilus islet genes (Fig 1).
When the allelic variant was expressed in T4ΔstkP (T4ΔstkPrXST) there was also a drop in ex-
pression of the pilus islet genes but not to the same level as that seen in T4ΔstkPrST.
Western blot analysis was also performed to evaluate if the change in gene expression ob-
served correlated to changes in total protein level. Changes in protein levels were evaluated in
T4ΔstkP, T4ΔstkPrST, T4ΔstkPrXST, T4ΔrrgB and TIGR4 for the pilus adhesin RrgA (Fig 2)
and the major pilin RrgB (Fig 3), which are key for adhesion to host cells and to pilus assembly
respectively [12,53]. Blots for both RrgA and RrgB show smears due to the majority of both
proteins being found as polymers. The single low molecular weight band shows the monomeric
form of the proteins. A large increase in RrgA protein levels in T4ΔstkP compared to TIGR4
was observed. When StkP (T4ΔstkPrST) was complemented there was a large decrease in
RrgA protein levels back to levels similar to those in TIGR4. This was not observed when the
allelic variant of StkP was expressed in T4ΔstkP (T4ΔstkPrXST) and only a small drop in
RrgA protein levels was observed compared to T4ΔstkP.

Fig 1. Pilus gene expression in T4ΔstkP and StkP complemented strains. Graph shows RT-PCR
expression of the genes present on the whole pilus islet (rlrA, rrgA, rrgB, rrgC, srtB, srtC, srtD) in T4ΔstkP,
T4ΔstkPrST and T4ΔstkPrXST compared to TIGR4. Fold change represents that of the mutant strain
compared to TIGR4. Each bar represents the average of three replicas and errors bars the
standard deviation.
doi:10.1371/journal.pone.0127212.g001

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 6 / 17


Regulation of the Pneumococcal Pilus

Fig 2. RrgA protein expression in T4ΔstkP and StkP complemented strains. Western blotting analysis was performed on TIGR4, T4ΔstkP,
T4ΔstkPrST, T4ΔstkPrXST and T4ΔrrgB at OD 0.6 (OD600nm) looking at expression of RrgA in all strains (α-RrgA antibody). Equal protein loading was
confirmed by equal expression of GroEL (α-GroEL antibody).
doi:10.1371/journal.pone.0127212.g002

RrgB protein levels were not clearly different in the kinase knockout compared to TIGR4.
However when complementing this mutant (T4ΔstkPrST) there was a clear drop in RrgB pro-
tein levels compared to TIGR4 and T4ΔstkP suggesting that overexpression of the kinase sup-
presses RrgB expression. When complementing this mutant with the allelic variant of StkP

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 7 / 17


Regulation of the Pneumococcal Pilus

Fig 3. RrgB protein expression in T4ΔstkP and StkP complemented strains. Western blotting analysis was performed on TIGR4, T4ΔstkP,
T4ΔstkPrST, T4ΔstkPrXST and T4ΔrrgB at OD 0.6 (OD600nm) looking at expression of RrgB in all strains (α-RrgB antibody). Equal protein loading was
confirmed by equal expression of GroEL (α-GroEL antibody).
doi:10.1371/journal.pone.0127212.g003

(T4ΔstkPrXST) the drop in RrgB levels was not as prominent but there was still a decrease
compared to TIGR4 and T4ΔstkP.
We observed several discrepancies between gene expression data and levels of protein ob-
served by western blot. This is presumably related to the different rates of accumulation of
pilins. Very little is known about regulation of stability and degradation of these proteins.

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 8 / 17


Regulation of the Pneumococcal Pilus

However there is some indication that regulation also occurs at the protein level with RlrA and
RrgA interacting to modulate expression levels of the islet [27].

Co-regulation of the kinase and phosphatase


Western blot analysis was performed on strains to evaluate the protein levels of the kinase and
phosphatase (Fig 4). As expected no StkP was observed in T4ΔstkP. When T4ΔstkP was com-
plemented with wild type StkP the levels of StkP observed was similar to that of TIGR4 (Fig 4).
When the allelic variant was expressed in T4ΔstkP a smaller StkP band was observed due to the
deletion of the 3rd PASTA domain. This band was also less dense suggesting less StkP is present,
however this is possibly due to the reduced binding of the StkP antibody as the PASTA domains
are the immunogenic region of StkP [52,54]. This is further validated via RT-PCR analysis of
the expression levels of the kinase in T4ΔstkPrST and T4ΔstkPrXST which shows similar lev-
els of expression. Levels of the phosphatase are also similar in T4ΔstkP, T4ΔstkPrST,
T4ΔstkPrXST (S2 Fig).
stkP and phpP are found adjacent on the chromosome and their functions have been closely
linked [28,39,55]. Further StkP is required for localisation of PhpP to the midcell so deletion of
StkP will in turn affect PhpP activity [37]. Therefore it is not surprising that there is altered
protein level of the phosphatase in the kinase mutants. With all three mutants showing lower
amounts of PhpP than that seen in TIGR4. The levels are higher when the wild type kinase is
expressed in T4ΔstkP (T4ΔstkPrST) at an unlinked part of the chromosome [47]. This would
suggest this is not a direct polar effect of deletion of stkP in TIGR4. Rather it is a consequence
of altered StkP levels, which are closely regulated to levels of PhpP [39].

Fig 4. Stkp and PhpP protein expression in T4ΔstkP and StkP complemented strains. Western blotting
analysis was performed on TIGR4, T4ΔstkP, T4ΔstkPrST, T4ΔstkPrXST and T4ΔrrgB at OD 0.6 (OD600nm)
looking at expression of StkP and PhpP in all strains (α-StkP/ -PhpP antibody). Equal protein loading was
confirmed by equal expression of GroEL (α-GroEL antibody).
doi:10.1371/journal.pone.0127212.g004

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 9 / 17


Regulation of the Pneumococcal Pilus

Modulation of adherence by StkP in TIGR4


To evaluate the effect of altered pilus levels on adherence in the StkP mutant and comple-
mented strains, their ability to adhere to Human Brain Microvascular Endothelial cells
(HBMEC) and A549 (Human lung epithelial carcinoma cells) were assessed (Fig 5). These cell
lines can be used to model in vitro adherence of pneumococci to host cells during meningitis
and pneumonia respectively.
Deletion of the kinase gene caused an increase in adherence to both cell lines. Deletion of
RrgB in T4ΔstkP (T4ΔstkPΔrrgB) abolished this increase in adherence showing the effect is
solely due to an increase in pilus expression and not other adhesins. T4ΔstkPΔrrgB adherence
capabilities were not different to that of T4ΔrrgB and TIGR4.
When the wild type kinase was expressed in T4ΔstkP the adherence to both cell lines re-
verted to the levels seen with TIGR4. When the allelic variant of StkP was expressed in
T4ΔstkP there was a trend towards a drop in adherence to both cells lines but this was not sig-
nificant. Similar adherence patterns of T4ΔstkP were observed also to Detroit 562 cells (naso-
pharyngeal carcinoma cell line) (S3 Fig.).

Regulation of the pilus by StkP at the population level


StkP modulates pilus levels through expression changes, which in turn modulates adherence. It
is not known if this is due to varying the number of pili on a single cell or the number of cells
that have pili. Recent findings have shown that the number of pili-positive cells within a popu-
lation can vary, although currently no genes have been shown to modulate this outside the
pilus islet genes [26,27]. This biphasic pattern of expression of the pilus is mediated by expres-
sion of rlrA and the regulator protein RlrA is part of a positive feedback mechanism for pilus
expression in individual bacterial cells [26,27].
Flow cytometry was used to evaluate the biphasic expression of pili. To deduce the number
of cells in a growing population that had RrgB present on the cell surface the whole bacterial
population was stained with anti-capsular antibody and the proportion of those expressing
pilli were measured using an RrgB specific antibody (Fig 6).
TIGR4 bacteria were gated in the FACS using the anti-capsular antibody. Gating using the
anti-RrgB fluorescence channel showed that 88% of the cells within this population were RrgB
positive. In T4ΔstkP the proportion increased to 99% of cells being pili positive. When T4ΔstkP
was complemented with StkP (T4ΔstkPrST) the proportion of pili-positive cells decreased,
with 26% of bacteria in the capsule positive population also being RrgB positive. When the alle-
lic variant of StkP (T4ΔstkPrXST) was expressed 51% of cells were RrgB positive. These data
suggest that StkP modulates the pilus at the population level.

Discussion
In this study we show that StkP regulates expression of the pneumococcal pilus in TIGR4. We
have shown that in strain TIGR4 the pilus modulates most of the adherence to human cells
grown in culture and that the level of adhesion is mediated through the kinase StkP.
The pneumococcal pilus has previously been shown to promote adherence to A549 cells.
Here we show it is also important for adherence to HBMEC cells suggesting a broad role of the
pilus in adherence to different host cells [12,25,53]. Specifically we suggest from our findings
that the pilus may aid adherence to brain endothelial cells during meningitis. This has been
shown in Streptococcus agalactiae (GBS) where the pilus plays a role in adherence to HBMEC
cells and has also been shown to aid penetration of the blood brain barrier [56,57]. However it
is difficult to determine the effect of pilus expression in the stkP knockout on virulence as it
causes other deleterious phenotypes, such as defects in cell division [29].

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 10 / 17


Regulation of the Pneumococcal Pilus

Fig 5. Adherence of T4ΔstkP and StkP complemented strains to different cell lines. Adherence of
strains TIGR4, T4ΔstkP, T4ΔstkPΔrrgB, T4ΔstkPrST, T4ΔstkPrXST and T4ΔrrgB was assessed to HBMEC
(A) and A549 (B) cell lines. Data is represented as percentage adherence relative to that of TIGR4 (100%,
dashed line), each bar is an average of three replicas and the error bars represent the standard error of the
mean. Statistical analysis was performed using a 1-way ANOVA with a Tukeys testing correction, * P<0.05. *
above the bar represent statistical significance compared to TIGR4 (not represented as a bar on the graphs).
doi:10.1371/journal.pone.0127212.g005

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 11 / 17


Regulation of the Pneumococcal Pilus

Fig 6. RrgB surface expression in T4ΔstkP and StkP complemented strains. Flow cytometry was
performed on T4ΔstkP, TIGR4, T4ΔstkPrST, T4ΔstkPrXST, T4ΔstkPΔrrgB and T4ΔrrgB. All samples were
initially gated on for being capsule positive (data not shown). This population was then gated on for RrgB
positive. Histograms show negative (left) and positive (right) RrgB populations in each strain. Table shows
the percentage RrgB positive and negative cells in a growing bacterial population.
doi:10.1371/journal.pone.0127212.g006

The role of the pilus in disease is still debated as presence of the pilus genes has not been
linked to invasive disease or outbreak strains [58]. However the pilus locus has been shown to
be more prevalent in the PCV7 serotypes and its re-emergence has been noted in Massachu-
setts in non-vaccine serotypes and is therefore thought to confer some advantage to these
strains [58,59]. There is evidence to suggest that the pilus provides an advantage during coloni-
sation due to its adhesive properties and its presence in strains has been shown to inversely cor-
relate to carriage of Staphylococcus aureus [60]. Here we show that the pilus adhesin allows
binding to endothelial and epithelial cells suggesting the role of the pilus is to aid colonisation.
Once invasion of normally sterile sites occurs (meninges/ lungs) the pilus can also adhere to
the cell types present within these niches. This is supported by the finding that the pilus adhe-
sin RrgA shares homology to eukaryotic like integrin domains which are involved in cell at-
tachment to extracellular matrix molecules (ECM) [61]. This suggests that these may be the
target of RrgA.
Recent studies have shown that the pilus displays a biphasic pattern of expression. In
TIGR4 30% of cells express the pilus on the cell surface however this varies between serotypes
[26,27]. In our experiments using TIGR4, 88% of the bacterial population expressed the pilus.
This difference may be due to differences in the TIGR4 strains used in different laboratories.
It has been shown that there are no gene expression differences between pili positive and
negative bacteria other than the genes present on the pilus islet itself [26]. This biphasic pattern
of expression has been observed in other Streptococci with Streptococcus pyogenes modulating
pilus expression at the population level based on temperature [62]. This phenomenon in the
pneumococcus is in part modulated by the binding of RrgA (adhesin) to RlrA (positive regula-
tor of the islet), which prevents RlrA positively regulating expression of the pilus islet [27].
Little is currently known about pilus biogenesis and assembly kinetics, which may explain
why in some instances we observe differences in gene expression and protein levels of the pilins
in the kinase knockout and complemented strains.
A large increase in adherence of TIGR4 to both HBMEC (7.5 fold) and A549 (15 fold) was
observed when StkP was deleted. Due to the fact there are only 11% more cells in the popula-
tion that are pili positive this increase seems disproportionate. However the recent structure of

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 12 / 17


Regulation of the Pneumococcal Pilus

the pilus shows that RrgA can also multimerise forming chains of the adhesin reaching beyond
the RrgB backbone [61]. The fact we see a large increase in RrgA protein levels in the StkP mu-
tants might suggest more branched pili are present which may increase the adhesive capabili-
ties of the bacterial cells. This is supported by the fact the amount of RrgA protein in the
different strains as determined by Western blotting directly correlated to the adherence capa-
bilities of the strains.
The kinase is highly conserved within the pneumococcus and shows 99.5% amino acid se-
quence homology between strains [52]. Here we identified an allelic variant of the kinase,
which is not present in any of the current genome sequenced strains [52]. Based on the fact the
kinase acts as a repressor of the pilus, when expressing the allelic variant this repression was
not as strong when the 3rd PASTA domain is not present. Whether this deletion alters StkP
function due to reduced activation of the kinase by a reduced ability to bind peptidoglycan pre-
cursors or by physically reducing the size of the extracellular domain that may reduce the prox-
imity to its substrate would require further study.
The mechanism by which the kinase modulates pilus expression is still unclear but a hy-
pothesis can be formed based on previous literature. The kinase has previously been shown to
phosphorylate two pneumococcal response regulators, one being the orphan response regula-
tor (SP_0376) and the other being the response regulator of TCS06 (SP_2193) [41,42]. RR06
has previously been shown to directly regulate the pneumococcal pilus through binding to the
promoter region of rlrA [23]. Therefore it is possible that deletion of the kinase affects the
phosphorylation state of RR06, which in turn alters pilus expression levels. In other bacteria
there are numerous references to the interaction of serine threonine kinases phosphorylating
RR of TCS pairs, which do so on a serine or threonine residue rather than the traditional aspar-
tate residue, which the cognate HK phosphorylates [63–65].
In summary this study has shown that the single serine threonine protein kinase regulates
the pneumococcal pilus in a serotype 4 and modulates adherence to HBMEC cells. This sug-
gests that the pneumococcal type 1 pilus may play a role in meningitis as well as its already de-
fined role in pneumonia and colonisation [12,23,53]. The study has also identified a new mode
of regulation at the population level of the kinase and has identified the kinase as the first gene
to modulate this type of expression for the pneumococcal pilus.

Supporting Information
S1 Fig. PASTA domains in StkP. Diagram shows the amino acid sequence of the extracellular
PASTA domains present at the C-terminal end of StkP. A- shows the four extracellular PASTA
domains present in TIGR4 StkP. In dark blue are the two 10 amino acid repeats present at the
end of the 2nd and 3rd PASTA domain, where the recombination event occurred in a serotype 4
strain (Xen35) containing the stkP allelic variant. Amino acids in red represent the amino acids
deleted in the StkP allelic variant. B- shows the StkP amino acid sequence in Xen 35, with the
3rd PASTA domain not present, and in dark blue the remaining 10 amino acid repeat.
(TIF)
S2 Fig. stkP and phpP expression in T4ΔstkP and StkP complements. A- Graph shows
RT-PCR expression of phpP in T4ΔstkP, T4ΔstkPrST and T4ΔstkPrXST compared to
TIGR4. Fold change represents that of the mutant strain compared to TIGR4. Each bar repre-
sents the average of three replicas and errors bars the standard deviation. B- Shows RT-PCR ex-
pression of stkP in T4ΔstkPrXST compared to T4ΔstkPrST. Fold change represents that of
T4ΔstkPrXST compared to T4ΔstkPrST. Each bar represents the average of three replicas
and errors bars the standard deviation.
(TIF)

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 13 / 17


Regulation of the Pneumococcal Pilus

S3 Fig. Adherence of T4ΔstkP to Detroit 562 cells. Adherence of strains TIGR4, T4ΔstkP,
T4ΔstkPΔrrgB and T4ΔrrgB was assessed to Detroit 562 cell lines. Data is represented as per-
centage adherence relative to that of TIGR4 (100%, dashed line). Each bar is an average of at
least two replicates and the error bars represent the standard error of the mean. Statistical anal-
ysis was performed using a 1-way ANOVA with a Tukeys testing correction,  P<0.01.
(TIF)
S1 Table. Strains and plasmids used in this study
(PDF)
S2 Table. Primers and related information used for construction of knockout strains via in
vitro mariner mutagenesis and splice overlap PCR and primers used to construct plasmids
pCP2 ST and pCP2 XST.
(PDF)
S3 Table. Primers and related information used for RT-PCR analysis of pilus, stkP and
phpP expression in T4ΔstkP, T4ΔstkPrST and T4ΔstkPrXST.
(PDF)

Acknowledgments
The StkP and PhpP antibodies were kindly provided by Dr Vijay Pancholi, Ohio State College
of Medicine. The RrgA antibody was kindly provided by Dr Markus Hilleringmann, Munich
University of Applied Sciences. For construction of the complemented strains plasmid pCEP2
was kindly provided by Professor Claverys, Université Paul Sabatier, Toulouse. HBMEC cells
were kindly provided by Professor Kwang Sik Kim, School of Medicine, Johns Hopkins, Uni-
versity, Baltimore, Maryland.

Author Contributions
Conceived and designed the experiments: TJM AMM JAH. Performed the experiments: JAH.
Analyzed the data: TJM AMM JAH. Wrote the paper: TJM AMM JAH.

References
1. Regev-Yochay G, Raz M, Dagan R, Porat N, Shainberg B, Pinco E, et al. Nasopharyngeal Carriage of
Streptococcus pneumoniae by Adults and Children in Community and Family Settings. Clin Infect Dis.
2004: 632–639. PMID: 14986245
2. O’Brien KL, Wolfson LJ, Watt JP, Henkle E, Deloria-Knoll M, McCall N, et al. Burden of disease caused
by Streptococcus pneumoniae in children younger than 5 years: global estimates. The Lancet. 2009;
374: 893–902. doi: 10.1016/S0140-6736(09)61204-6 PMID: 19748398
3. Henrichsen J. Six newly recognized types of Streptococcus pneumoniae. Journal of Clinical Microbiolo-
gy. 1995; 33: 2759–2762. PMID: 8567920
4. Hicks LA, Harrison LH, Flannery B, Hadler JL, Schaffner W, Craig AS, et al. Incidence of pneumococcal
disease due to non-pneumococcal conjugate vaccine (PCV7) serotypes in the United States during the
era of widespread PCV7 vaccination, 1998–2004. J Infect Dis. 2007; 196: 1346–1354. PMID:
17922399
5. Spratt BG, Greenwood BM. Prevention of pneumococcal disease by vaccination: does serotype re-
placement matter? The Lancet. 2000; 356: 1210–1211.
6. Coffey TJ, Daniels M, Enright MC, Spratt BG. Serotype 14 variants of the Spanish penicillin- resistant
serotype 9V clone of Streptococcus pneumoniae arose by large recombinational replacements of the
cpsA-pbpla region. Microbiology. 1999; 145: 2023–2031. PMID: 10463168
7. Coffey TJ, Dowson CG, Daniels M, Zhou J, M C, Spratt BG, et al. Horizontal transfer of multiple penicil-
lin-binding protein genes, and capsuiar biosynthetic genes, in natural populations of Streptococcus
pneumoniae. Molecular Microbiology. 1991; 5: 2255–2260. PMID: 1766389

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 14 / 17


Regulation of the Pneumococcal Pilus

8. Coffey TJ, Enright MC, Daniels M, Morona JK, Morona R, Hryniewicz W, et al. Recombinational ex-
changes at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among
natural isolates of Streptococcus pneumoniae. Molecular Microbiology. 1998; 27: 73–83. PMID:
9466257
9. Mitchell AM, Mitchell TJ. Streptococcus pneumoniae: virulence factors and variation. Clinical Microbiol-
ogy and Infection. 2010; 16: 411–418. doi: 10.1111/j.1469-0691.2010.03183.x PMID: 20132250
10. Moschioni M, Donati C, Muzzi A, Masignani V, Censini S, Hanage WP, et al. Streptococcus pneumo-
niae contains 3 rlrA pilus variants that are clonally related. J Infect Dis. 2008; 197: 888–896. doi: 10.
1086/528375 PMID: 18269316
11. Bagnoli F, Moschioni M, Donati C, Dimitrovska V, Ferlenghi I, Facciotti C, et al. A second pilus type in
Streptococcus pneumoniae is prevalent in emerging serotypes and mediates adhesion to host cells. J
Bacteriol. 2008; 190: 5480–5492. doi: 10.1128/JB.00384-08 PMID: 18515415
12. Barocchi MA, Ries J, Zogaj X, Hemsley C, Albiger B, Kanth A, et al. A pneumococcal pilus influences
virulence and host inflammatory responses. Proc Natl Acad Sci U S A. 2006; 103: 2857–2862. PMID:
16481624
13. Hava DL, Hemsley CJ, Camilli A. Transcriptional Regulation in the Streptococcus pneumoniae rlrA
Pathogenicity Islet by RlrA. Journal of Bacteriology. 2003; 185: 413–421. PMID: 12511486
14. Hava DL, Hemsley CJ, Camilli A. Transcriptional regulation in the Streptococcus pneumoniae rlrA path-
ogenicity islet by RlrA. J Bacteriol. 2003; 185: 413–421. PMID: 12511486
15. Munoz-Elias EJ, Marcano J, Camilli A. Isolation of Streptococcus pneumoniae biofilm mutants and their
characterization during nasopharyngeal colonization. Infect Immun. 2008; 76: 5049–5061. doi: 10.
1128/IAI.00425-08 PMID: 18794289
16. Moschioni M, De Angelis G, Harfouche C, Bizzarri E, Filippini S, Mori E, et al. Immunization with the
RrgB321 fusion protein protects mice against both high and low pilus-expressing Streptococcus pneu-
moniae populations. Vaccine. 2012; 30: 1349–1356. doi: 10.1016/j.vaccine.2011.12.080 PMID:
22210141
17. Hava DL, Camilli A. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence fac-
tors. Molecular Microbiology. 2002; 5: 1389–1405. PMID: 12207705
18. Sebert ME, Palmer LM, Rosenberg M, Weiser JN. Microarray-Based Identification of htrA, a Strepto-
coccus pneumoniae Gene That Is Regulated by the CiaRH Two-Component System and Contributes
to Nasopharyngeal Colonization. Infection and Immunity. 2002; 70: 4059–4067. PMID: 12117912
19. Basset A, Zhang F, Benes C, Sayeed S, Herd M, Thompson C, et al. Toll-like receptor (TLR) 2 mediates
inflammatory responses to oligomerized RrgA pneumococcal pilus type 1 protein. J Biol Chem. 2013;
288: 2665–2675. doi: 10.1074/jbc.M112.398875 PMID: 23233677
20. Orrskog S, Rounioja S, Spadafina T, Gallotta M, Norman M, Hentrich K, et al. Pilus adhesin RrgA inter-
acts with complement receptor 3, thereby affecting macrophage function and systemic pneumococcal
disease. MBio. 2012; 4: e00535–00512. doi: 10.1128/mBio.00535-12 PMID: 23269830
21. Haas W, Sublett J, Kaushal D, Tuomanen EI. Revising the role of the pneumococcal vex-vncRS locus
in vancomycin tolerance. J Bacteriol. 2004; 186: 8463–8471. PMID: 15576796
22. Hendriksen WT, Silva N, Bootsma HJ, Blue CE, Paterson GK, Kerr AR, et al. Regulation of gene ex-
pression in Streptococcus pneumoniae by response regulator 09 is strain dependent. J Bacteriol. 2007;
189: 1382–1389. PMID: 17085554
23. Rosch JW, Mann B, Thornton J, Sublett J, Tuomanen E. Convergence of regulatory networks on the
pilus locus of Streptococcus pneumoniae. Infect Immun. 2008; 76: 3187–3196. doi: 10.1128/IAI.
00054-08 PMID: 18443093
24. Song XM, Connor W, Hokamp K, Babiuk LA, Potter AA. The growth phase-dependent regulation of the
pilus locus genes by two-component system TCS08 in Streptococcus pneumoniae. Microb Pathog.
2009; 46: 28–35. doi: 10.1016/j.micpath.2008.10.006 PMID: 18983906
25. Hemsley C, Joyce E, Hava DL, Kawale A, Camilli A. MgrA, an Orthologue of Mga, Acts as a Transcrip-
tional Repressor of the Genes within the rlrA Pathogenicity Islet in Streptococcus pneumoniae. Journal
of Bacteriology. 2003; 185: 6640–6647. PMID: 14594838
26. De Angelis G, Moschioni M, Muzzi A, Pezzicoli A, Censini S, Delany I, et al. The Streptococcus pneu-
moniae pilus-1 displays a biphasic expression pattern. PLoS One. 2011; 6: e21269. doi: 10.1371/
journal.pone.0021269 PMID: 21731688
27. Basset A, Turner KH, Boush E, Sayeed S, Dove SL, Malley R. Expression of the type 1 pneumococcal
pilus is bistable and negatively regulated by the structural component RrgA. Infect Immun. 2011; 79:
2974–2983. doi: 10.1128/IAI.05117-11 PMID: 21576325

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 15 / 17


Regulation of the Pneumococcal Pilus

28. Pallova P, Hercik K, Saskova L, Novakova L, Branny P. A eukaryotic-type serine/threonine protein ki-
nase StkP of Streptococcus pneumoniae acts as a dimer in vivo. Biochem Biophys Res Commun.
2007; 355: 526–530. PMID: 17307148
29. Saskova L, Novakova L, Basler M, Branny P. Eukaryotic-type serine/threonine protein kinase StkP is a
global regulator of gene expression in Streptococcus pneumoniae. J Bacteriol. 2007; 189: 4168–4179.
PMID: 17416671
30. Echenique J, Kadioglu A, Romao S, Andrew PW, Trombe MC. Protein Serine/Threonine Kinase StkP
Positively Controls Virulence and Competence in Streptococcus pneumoniae. Infection and Immunity.
2004; 72: 2434–2437. PMID: 15039376
31. Yeats C, Finn RD, Bateman A. The PASTA domain: a β-lactam-binding domain. 2002; 27: 438–440.
32. Maestro B, Novakova L, Hesek D, Lee M, Leyva E, Mobashery S, et al. Recognition of peptidoglycan
and beta-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Strepto-
coccus pneumoniae. FEBS Lett. 2011; 585: 357–363. doi: 10.1016/j.febslet.2010.12.016 PMID:
21167155
33. Maurer P, Todorova K, Sauerbier J, Hakenbeck R. Mutations in Streptococcus pneumoniae penicillin-
binding protein 2x: importance of the C-terminal penicillin-binding protein and serine/threonine kinase-
associated domains for beta-lactam binding. Microb Drug Resist. 2012; 18: 314–321. doi: 10.1089/
mdr.2012.0022 PMID: 22455550
34. Gordon E, Mouz N, Duee E, Dideberg O. The crystal structure of the penicillin-binding protein 2x from
Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance. J Mol Biol. 2000;
299: 477–485. PMID: 10860753
35. Spratt BG. Properties of the Penicillin-Binding Proteins of Escherichia coli K 12. European journal Bio-
chemistry. 1977: 341–352.
36. Frere JM, Joris B. Penicillin-sensitive enzymes in peptidoglycan biosynthesis. Crit Rev Microbiol. 1985;
11: 299–394. PMID: 3888533
37. Beilharz K, Novakova L, Fadda D, Branny P, Massidda O, Veening JW. Control of cell division in Strep-
tococcus pneumoniae by the conserved Ser/Thr protein kinase StkP. Proc Natl Acad Sci U S A. 2012;
109: E905–913. doi: 10.1073/pnas.1119172109 PMID: 22431591
38. Giefing C, Jelencsics KE, Gelbmann D, Senn BM, Nagy E. The pneumococcal eukaryotic-type serine/
threonine protein kinase StkP co-localizes with the cell division apparatus and interacts with FtsZ in
vitro. Microbiology. 2010; 156: 1697–1707. doi: 10.1099/mic.0.036335-0 PMID: 20223804
39. Novakova L, Saskova L, Pallova P, Janecek J, Novotna J, Ulrych A, et al. Characterization of a eukary-
otic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and
identification of kinase substrates. FEBS J. 2005; 272: 1243–1254. PMID: 15720398
40. Novakova L, Bezouskova S, Pompach P, Spidlova P, Saskova L, Weiser J, et al. Identification of multi-
ple substrates of the StkP Ser/Thr protein kinase in Streptococcus pneumoniae. J Bacteriol. 2010;
192: 3629–3638. doi: 10.1128/JB.01564-09 PMID: 20453092
41. Ulijasz AT, Falk SP, Weisblum B. Phosphorylation of the RitR DNA-binding domain by a Ser-Thr phos-
phokinase: implications for global gene regulation in the streptococci. Mol Microbiol. 2009; 71: 382–
390. doi: 10.1111/j.1365-2958.2008.06532.x PMID: 19040630
42. Agarwal S, Agarwal S, Pancholi P, Pancholi V. Strain-specific regulatory role of eukaryote-like serine/
threonine phosphatase in pneumococcal adherence. Infect Immun. 2012; 80: 1361–1372. doi: 10.
1128/IAI.06311-11 PMID: 22311926
43. Lampe DJ, Akerley BJ, Rubin EJ, Mekalanos JJ, Robertson HM. Hyperactive transposase mutants of
the Himar1 mariner transposon. Proc Natl Acad Sci USA. 1999; 96: 11428–11433. PMID: 10500193
44. Lampe DJ, Churchill MEA, Robertson HM. A purified mariner transposase is sufficient to mediate trans-
position in vitro. The EMBO Journal. 1996; 15: 5470–5479. PMID: 8895590
45. Mann B, Orihuela C, Antikainen J, Gao G, Sublett J, Korhonen TK, et al. Multifunctional role of choline
binding protein G in pneumococcal pathogenesis. Infect Immun. 2006; 74: 821–829. PMID: 16428724
46. Sung CK, Li H, Claverys JP, Morrison DA. An rpsL cassette, janus, for gene replacement through nega-
tive selection in Streptococcus pneumoniae. Appl Environ Microbiol. 2001; 67: 5190–5196. PMID:
11679344
47. Guiral S, Henard V, Laaberki MH, Granadel C, Prudhomme M, Martin B, et al. Construction and evalua-
tion of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Strep-
tococcus pneumoniae. Microbiology. 2006; 152: 343–349. PMID: 16436422
48. Blue CE, Mitchell TJ. Contribution of a Response Regulator to the Virulence of Streptococcus pneumo-
niae Is Strain Dependent. Infection and Immunity. 2003; 71: 4405–4413. PMID: 12874319
49. Livak KJ, Schmittgen TD. Analaysis of relative gene expression data using real-time quantitative PCR
and the 2)-delta Delta (CT) method. Methods Enzymol. 2001; 24: 402–408.

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 16 / 17


Regulation of the Pneumococcal Pilus

50. Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, et al. Complete genome sequence
of a virulent isolate of Streptococcus pneumoniae. Science. 2001; 293: 498–506. PMID: 11463916
51. Aaberge IS, Engz J, Lermark G, Levik M. Virulence of Streptococcuspneumoniaein mice: a standard-
ized method for preparation and frozen storage of the experimental bacterial inoculum. Microbial Patho-
genesis. 1995: 141–152. PMID: 7643743
52. Giefing C, Meinke AL, Hanner M, Henics T, Bui MD, Gelbmann D, et al. Discovery of a novel class of
highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with
human antibodies. J Exp Med. 2008; 205: 117–131. doi: 10.1084/jem.20071168 PMID: 18166586
53. Nelson AL, Ries J, Bagnoli F, Dahlberg S, Falker S, Rounioja S, et al. RrgA is a pilus-associated adhe-
sin in Streptococcus pneumoniae. Mol Microbiol. 2007; 66: 329–340. PMID: 17850254
54. Schmid P, Selak S, Keller M, Luhan B, Magyarics Z, Seidel S, et al. Th17/Th1 biased immunity to the
pneumococcal proteins PcsB, StkP and PsaA in adults of different age. Vaccine. 2011; 29: 3982–
3989. doi: 10.1016/j.vaccine.2011.03.081 PMID: 21481328
55. Osaki M, Arcondeguy T, Bastide A, Touriol C, Prats H, Trombe MC. The StkP/PhpP signaling couple in
Streptococcus pneumoniae: cellular organization and physiological characterization. J Bacteriol. 2009;
191: 4943–4950. doi: 10.1128/JB.00196-09 PMID: 19502404
56. Maisey HC, Hensler M, Nizet V, Doran KS. Group B streptococcal pilus proteins contribute to adher-
ence to and invasion of brain microvascular endothelial cells. J Bacteriol. 2007; 189: 1464–1467.
PMID: 17041051
57. Banerjee A, Kim BJ, Carmona EM, Cutting AS, Gurney MA, Carlos C, et al. Bacterial Pili exploit integrin
machinery to promote immune activation and efficient blood-brain barrier penetration. Nat Commun.
2011; 2: 462. doi: 10.1038/ncomms1474 PMID: 21897373
58. Basset A, Trzcinski K, Hermos C, O'Brien KL, Reid R, Santosham M, et al. Association of the pneumo-
coccal pilus with certain capsular serotypes but not with increased virulence. J Clin Microbiol. 2007;
45: 1684–1689. PMID: 17392439
59. Regev-Yochay G, Hanage WP, Trzcinski K, Rifas-Shiman SL, Lee G, Bessolo A, et al. Re-emergence
of the type 1 pilus among Streptococcus pneumoniae isolates in Massachusetts, USA. Vaccine. 2010;
28: 4842–4846. doi: 10.1016/j.vaccine.2010.04.042 PMID: 20434550
60. Regev-Yochay G, Lipsitch M, Basset A, Rubinstein E, Dagan R, Raz M, et al. The pneumococcal pilus
predicts the absence of Staphylococcus aureus co-colonization in pneumococcal carriers. Clin Infect
Dis. 2009; 48: 760–763. doi: 10.1086/597040 PMID: 19207082
61. El Mortaji L, Fenel D, Vernet T, Di Guilmi AM. Association of RrgA and RrgC into the Streptococcus
pneumoniae pilus by sortases C-2 and C-3. Biochemistry. 2012; 51: 342–352. doi: 10.1021/bi201591n
PMID: 22122269
62. Nakata M, Koller T, Moritz K, Ribardo D, Jonas L, McIver KS, et al. Mode of expression and functional
characterization of FCT-3 pilus region-encoded proteins in Streptococcus pyogenes serotype M49. In-
fect Immun. 2009; 77: 32–44. doi: 10.1128/IAI.00772-08 PMID: 18852238
63. Lin WJ, Walthers D, Connelly JE, Burnside K, Jewell KA, Kenney LJ, et al. Threonine phosphorylation
prevents promoter DNA binding of the Group B Streptococcus response regulator CovR. Mol Microbiol.
2009; 71: 1477–1495. doi: 10.1111/j.1365-2958.2009.06616.x PMID: 19170889
64. Jiang SM, Cieslewicz MJ, Kasper DL, Wessels MR. Regulation of virulence by a two-component sys-
tem in group B streptococcus. J Bacteriol. 2005; 187: 1105–1113. PMID: 15659687
65. Agarwal S, Agarwal S, Pancholi P, Pancholi V. Role of serine/threonine phosphatase (SP-STP) in
Streptococcus pyogenes physiology and virulence. J Biol Chem. 2011; 286: 41368–41380. doi: 10.
1074/jbc.M111.286690 PMID: 21917918

PLOS ONE | DOI:10.1371/journal.pone.0127212 June 19, 2015 17 / 17

You might also like

pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy