See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/50266849

Structural and Functional Characterization of the Streptococcus pneumoniae

RrgB Pilus Backbone D1 Domain

Article in Journal of Biological Chemistry · March 2011

DOI: 10.1074/jbc.M110.202739 · Source: PubMed

CITATIONS READS
23 181

15 authors, including:

Sara Melchiorre Carla Emolo

GlaxoSmithKline University of Chicago
8 PUBLICATIONS 159 CITATIONS 11 PUBLICATIONS 575 CITATIONS

SEE PROFILE SEE PROFILE

Maria Scarselli Werner Pansegrau

Novartis GlaxoSmithKline
139 PUBLICATIONS 8,699 CITATIONS 56 PUBLICATIONS 5,044 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Iron Sulfur cluster Proteins View project

Characterization of Clostridium difficile virulence factors View project

All content following this page was uploaded by Francesca Cantini on 02 June 2014.

The user has requested enhancement of the downloaded file.

 Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2011/03/02/M110.202739.DC1.html

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 16, pp. 14588 –14597, April 22, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Structural and Functional Characterization of the

Streptococcus pneumoniae RrgB Pilus Backbone D1 Domain*□ S

Received for publication, November 11, 2010, and in revised form, February 16, 2011 Published, JBC Papers in Press, March 2, 2011, DOI 10.1074/jbc.M110.202739
Maria Antonietta Gentile‡, Sara Melchiorre‡, Carla Emolo‡, Monica Moschioni‡, Claudia Gianfaldoni‡,
Laura Pancotto‡, Ilaria Ferlenghi‡, Maria Scarselli‡, Werner Pansegrau‡, Daniele Veggi‡, Marcello Merola‡§,
Francesca Cantini¶, Paolo Ruggiero‡, Lucia Banci¶1, and Vega Masignani‡2
From the ‡Novartis Vaccines and Diagnostics Research Center, Via Fiorentina 1, Siena 53100, Italy, the ¶Magnetic Resonance
Center, Department of Chemistry, University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy, and the §Università di
Napoli Federico II, Monte Sant’Angelo, Via Cintia, Napoli 80126, Italy

Streptococcus pneumoniae expresses on its surface adhesive and meningitis (1– 6). However, S. pneumoniae is also a com-
pili, involved in bacterial attachment to epithelial cells and vir- mon inhabitant of the respiratory tract of children and healthy

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

ulence. The pneumococcal pilus is composed of three proteins, adults. This carriage state could represent a risk factor for the
RrgA, RrgB, and RrgC, each stabilized by intramolecular isopep- development of respiratory diseases but also the source for
tide bonds and covalently polymerized by means of intermolec- pneumococcal transmission to other individuals (7–9). Like
ular isopeptide bonds to form an extended fiber. RrgB is the most streptococci, S. pneumoniae decorates its surface with
pilus scaffold subunit and is protective in vivo in mouse models long filaments known as pili (10 –14). Pneumococcal pili have
of sepsis and pneumonia, thus representing a potential vaccine previously been associated with virulence and the capability of
candidate. The crystal structure of a major RrgB C-terminal por- the microorganism to adhere better to epithelial cells and to
tion featured an organization into three independently folded colonize the nasopharynx (10, 15, 16). The pneumococcal pilus
protein domains (D2–D4), whereas the N-terminal D1 domain is a multimeric structure consisting of three proteins (RrgA,
(D1) remained unsolved. We have tested the four single recom- RrgB, and RrgC) polymerized by three sortase enzymes (SrtC1,
binant RrgB domains in active and passive immunization stud- SrtC2, and SrtC3) through the formation of covalent intermo-
ies and show that D1 is the most effective, providing a level of lecular isopeptide bonds (17–21). In particular, multiple copies
protection comparable with that of the full-length protein. To of RrgB are polymerized to form the scaffold of the pilus,
elucidate the structural features of D1, we solved the solution whereas the major adhesin, RrgA, and the putative anchor,
structure of the recombinant domain by NMR spectroscopy. The RrgC, are localized at the tip and at the base of the pilus, respec-
spectra analysis revealed that D1 has many flexible regions, does tively (15, 22, 23).
not contain any intramolecular isopeptide bond, and shares with Recently, the structure of a major portion of RrgB (residues
the other domains an Ig-like fold. In addition, we demonstrated, by 184 – 627) was solved at a 1.6 Å resolution (24) and revealed an
site-directed mutagenesis and complementation in S. pneumoniae, organization into three independently folded IgG-like domains
that the D1 domain contains the Lys residue (Lys-183) involved in (D2, D3, and D4, residues 184 –326, 326 – 446, and 446 – 627,
the formation of the intermolecular isopeptide bonds and pilus respectively). On the contrary, the structure of the RrgB N-ter-
polymerization. Finally, we present a model of the RrgB protein minal region (D1, residues 1–184), likely constituting a fourth
architecture along with the mapping of two surface-exposed linear independently folded domain, remained unsolved due to the
epitopes recognized by protective antisera. failure to obtain the crystals of the full-length (FL)3 RrgB (24).
Interestingly, each of the D2, D3, and D4 domains is stabilized
by one intramolecular isopeptide bond. These covalent link-
Streptococcus pneumoniae is an important human pathogen ages, formed between Lys and Asn residues, have been found in
responsible for diseases such as otitis media, pneumonia, sepsis, other pilus proteins (19, 25–28) and are thought to play a role
similar to that of disulfide bonds; they confer in fact a rigid
* This work was supported by Ministero dell’Istruzione, dell’Università e della molecular architecture to the pili and make them less suscepti-
Ricerca (Fondi per gli Investimenti della Ricerca di Base-Proteomica ble to proteolytic cleavage (Fig. 1).
RBRN07BMCT) and Integrated Structural Biology Infrastructure for Europe
Contract 211252. Because some of the authors are employees of Novartis In the pilus backbone assembly RrgB molecules are linked
Vaccines, there are competing financial interests. together by sortases through intermolecular isopeptide bond
□S
The on-line version of this article (available at http://www.jbc.org) contains formation between a Thr within the C-terminal LPXTG motif
Materials and Methods, supplemental Tables S1–S6, and supplemental
Figs. S1–S6.
of a molecule and a Lys located at the N terminus of the follow-
The atomic coordinates and structure factors (code 2L40) have been deposited in ing molecule (18, 26, 29) (Fig. 1). In Corynebacterium diphthe-
the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, riae, where the general principles of pilus assembly were first
Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
The NMR chemical shifts have been deposited in the BioMagResBank (acces-
3
sion no. 17246). The abbreviations used are: FL, full-length; BisTris, bis(2-hydroxyethyl)imi-
1
To whom correspondence may be addressed. Tel.: 39-055-4574263; Fax: notris(hydroxymethyl)methane; cryo-EM, cryoelectron microscopy; cfu,
39-055-4574253; E-mail: [email protected]. colony-forming units; HSQC, heteronuclear single quantum coherence;
2
To whom correspondence may be addressed. Tel.: 39-0577-243319; Fax: PDB, Protein Data Bank; r.m.s.d., root mean square deviation; WB, Western
39-0577-243564; E-mail: [email protected]. blot.

14588 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 16 • APRIL 22, 2011
 Protective RrgB D1 Domain Involved in Pilus Polymerization
RrgB or RrgB mutated forms was under control of the erythro-
mycin constitutive promoter (Pc) which was amplified with the
primers listed in supplemental Table S2 and cloned immedi-
ately upstream rrgB (EcoRI, BamHI). All plasmids were con-
firmed by sequencing.
Generation of rrgB Deletion Mutants and rrgB Comple-
FIGURE 1. Schematic representation of pilus backbone protein RrgB. Pilus mentants—A TIGR4 ⌬rrgB isogenic mutant was generated by
scaffold is composed by multiples copies of RrgB protein in a head-to-tail
arrangement. Pilus polymerization occurs through intermolecular isopeptide
allelic exchange. Fragments of ⬃500 bp upstream and down-
bonds (red), whereas each RrgB protein is stabilized by intramolecular isopep- stream the target gene were amplified by PCR (oligonucleotides
tide bonds (black). Lys-183 as a residue involved in the intermolecular bond are listed in supplemental Table S2) and spliced into a kanamy-
has been identified in the present work.
cin resistance cassette by using overlap extension PCR; the PCR
fragments were then cloned into pGEMt (Promega) and trans-
established, the functional Lys is located within a conserved formed in S. pneumoniae with conventional methods (35). To
YPKN “pilin” motif (18, 27, 30). Nevertheless, this sequence select the bacteria in which the target gene was replaced with
is not absolutely required for polymerization as demon- the resistance cassette, bacteria were plated on blood-agar
strated by studies on the Spy0128 pilin of Streptococcus pyo- plates with kanamycin (500 ␮g/ml). The presence of the iso-

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

genes, where the lysine forming the intermolecular isopep- genic mutation was confirmed by PCR and Western blot (WB)
tide bond and responsible for pilus polymerization is located analysis. To obtain RrgB-complemented mutants, pMU1328
into 159GSKVPI164 motif even though the YPKN pilin motif plasmids containing WT rrgB or rrgB mutated forms were
is also present (26, 31). transformed into TIGR4 ⌬rrgB with conventional methods.
RrgB, along with the other two pilus proteins RrgA and RrgC, Transformants selection was performed by supplementing
was previously shown to confer protection in mouse models of media with kanamycin (500 ␮g/ml) and erythromycin (1
infection and therefore is regarded as a potential candidate for a ␮g/ml). The complemented mutants were then analyzed by
new generation of protein-based vaccines (32, 33). We have PCR; expression of FL WT RrgB or RrgB mutants was detected
investigated the protective ability of the single recombinant D1, by WB analysis of whole cell lysates.
D2, D3, and D4 domains of RrgB in a mouse model of sepsis,
SDS-PAGE and Western Blot Analysis—SDS-PAGE analysis
and here we provide evidence that D1 is the most protective,
was performed using NuPAGETM 4 –12% BisTris gradient gels
followed by D4. Furthermore, we present the solution structure
(Invitrogen) according to the manufacturer’s instructions.
of the recombinant D1 obtained by NMR spectroscopy and
Hi-MarkTM prestained HMW (Invitrogen) served as protein
show that Lys-183 of D1 is engaged in the intermolecular iso-
standard. Gels were processed for WB analysis by using stan-
peptide bond formation during pilus polymerization. Finally,
dard protocols. Mouse polyclonal antibodies raised against
we propose a possible model of the entire RrgB molecule and
recombinant His-tagged RrgB were used at 1/3000 dilution.
show the positions of two linear epitopes possibly involved in
Secondary goat anti-mouse IgG alkaline phosphatase-conju-
the protection mechanism.
gated antibodies (Promega) were used at 1/5000, and signals
MATERIALS AND METHODS were developed by using Western Blue Stabilized Substrate for
Bacterial Strains and Cultures—For the animal experiments, Alkaline Phosphatase (Promega).
the S. pneumoniae TIGR4 (serotype 4) strain was used. Bacteria Animal Experiments—Animal studies were done in compli-
were grown, frozen in aliquots at ⫺80 °C, and titrated as already ance with the current law approved by the local Animal Ethics
reported (32). Immediately prior to challenge, frozen aliquots Committee and authorized by the Italian Ministry of Health.
were thawed and diluted in PBS to reach the working Female, 6-week-old, specific pathogen-free BALB/c mice
concentration. (Charles River) received three intraperitoneal immunizations, 2
Cloning and Protein Expression and Purification—Standard weeks apart. Each dose was composed of 20 ␮g of either the
recombinant DNA techniques were used to construct the single RrgB domains or the FL RrgB, or of a combination of the
expression plasmids (pET21b⫹; Novagen) and to express and four RrgB domains (D1⫹D2⫹D3⫹D4), 10 ␮g each, along with
purify the recombinant C-terminal His-tagged proteins (for 400 ␮g of aluminum hydroxide as an adjuvant, in a final volume
details, see supplemental Materials and Methods, and the prim- of 200 ␮l of PBS. Control animals received the same course of
ers used are listed in supplemental Table S1). The affinity-pu- saline plus adjuvant. Ten days after the third immunization,
rified proteins were subsequently used to immunize CD1 mice samples of sera were obtained from each animal and pooled
or rabbits for antibody generation (Charles River Laboratory) according to the immunization group to be used in passive
and BALB/c mice to evaluate the protective efficacy. serum transfer experiment. Two weeks after the third immuni-
Complementation Plasmids—Wild-type or mutant rrgB zation, each mouse was challenged intraperitoneally with a
genes were amplified from chromosomal DNA of TIGR4 strain mean dose of 1.6 ⫻ 102 cfu of TIGR4. Bacteremia was evaluated
by PCR by using the primers listed in supplemental Table S2; 24 h after challenge, and mortality course monitored for 10 days
point mutations were introduced by overlap extension PCR by after challenge as already reported (32). The animals were
using specific primers (supplemental Table S2). PCR products euthanized when they exhibited defined humane end points
were then cloned into the complementation plasmid pMU1328 that had been preestablished for the study in agreement with
between the BamHI and SalI restriction sites (34). Expression of Novartis Animal Welfare Policies.

APRIL 22, 2011 • VOLUME 286 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 14589
Protective RrgB D1 Domain Involved in Pilus Polymerization
For passive protection experiments, 8-week-old mice were Ha chemical shifts, using TALOS⫹ (36). Distance constraints
used. Fifteen minutes before TIGR4 intraperitoneal challenge for structure determination were obtained from 15N-edited and
(102 cfu/mouse), each mouse received intraperitoneally 50 ␮l of 13
C-edited three-dimensional NOESY-HSQC. 3131 meaning-
pooled mouse sera against recombinant D1 or D4, or of control ful proton-proton distance restraints (supplemental Table S4),
sera obtained immunizing with adjuvant plus saline. with 114 ␸ and 120 ␺ backbone dihedral angles restraints were
Statistical Analysis—Bacteremia and mortality course were included in structure calculations. The exchangeability of the
analyzed by the Mann-Whitney U test. Survival rates were ana- backbone amide hydrogen nuclei with solvent protons was
lyzed by Fisher’s exact test. One-tailed or two-tailed tests were investigated through a 1H-15N HSQC experiment performed
used to compare immunized groups with the control group or on a protein sample dialyzed against deuterated buffer for 3
each other, respectively. Values of p ⱕ 0.05 were considered days. Hydrogen bond constraints for the slowly deuterium-ex-
and referred to as significant. Values of p ⱕ 0.1 were referred to changing amide protons of the ␤-strands were introduced at
as a trend. later steps of the structure calculations.
Flow Cytometry on Entire Bacteria—TIGR4 were grown in Structure calculations were performed through iterative
Todd-Hewitt yeast extract broth to an exponential phase cycles of CYANA-2.1 (37) followed by restrained energy mini-
(A600 ⫽ 0.25), fixed with 2% formaldehyde, and then stained mization with the AMBER 10.0 Package in explicit water sol-
with pooled mouse antisera raised against FL RrgB or RrgB vent (38). The quality of the structures was evaluated by using

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

domains at 1:400 dilution. Mouse IgG were detected with the iCING validation program (for details, see supplemental
FITC-conjugated goat anti-mouse IgG (Jackson Laboratories) Table S4). The program MOLMOL was subsequently used for
at 1:100 dilution, and bacterial staining was analyzed by using a structure analysis (39).
FACS-Calibur cytometer (Becton Dickinson). Sera from mice The final bundle of 20 conformers of D1 has an average target
immunized with PBS plus adjuvant were used as negative function of 1.36 ⫾ 0.13 (CYANA units). The average backbone
control. r.m.s.d. value (over residues 28 –183) is 0.71 ⫾ 0.19Å, and the
ELISA—96-well MaxiSorpTM flat-bottom plates (Nunc) were all-heavy atoms r.m.s.d. value is 0.96 ⫾ 0.16. Per-residue
coated with 0.2 ␮g/well FL RrgB overnight at 4 °C. Plates were r.m.s.d. values are shown in supplemental Fig. S1.
then washed three times with PBS/0.05% Tween 20 and satu- Heteronuclear Relaxation Data—The dynamic properties of
rated for 1 h at 37 °C with PBS/1% BSA. Following three wash- D1 have been characterized experimentally through 15N relax-
ing steps with PBS/0,05% Tween 20, the plates were incubated ation measurements. 15N longitudinal and traverse relaxation
for 2 h at 37 °C with serial dilutions of the pooled mouse sera. rates (40) and 15N{1H} NOEs (41) were recorded at 298 K at 500
After another three washing steps, bound antigen-specific MHz, using a protein concentration of 0.8 mM.
mouse IgGs were revealed with alkaline phosphatase-conju- The average backbone 15N longitudinal R1 and transversal R2
gated goat anti-mouse IgG (Sigma), followed by the phospha- relaxation rates and 15N{1H} NOE values are 1.45 ⫾ 0.1 s⫺1,
tase alkaline substrate p-nitrophenyl phosphate (Sigma). The 16.18 ⫾ 1.5 s⫺1, and 0.71 ⫾ 0.04, respectively (supplemental
intensity of color was quantified with an ELISA plate reader at Fig. S2). They are essentially homogeneous along the entire
A405. The antibody titer was expressed as the log10 of the recip- polypeptide sequence, with the exception of residues located at
rocal of the serum dilution that gave A405 ⫽ 1. the C and N termini and three loop regions (56 – 69, 148 –162,
PepScan Analysis—Arrayed peptides were synthesized in and 173–177). The correlation time for the molecule tumbling
situ on glass fiber membranes. Membranes were conditioned (␶c), as estimated from the R2/R1 ratio, is 11.49 ⫾ 1.9 ns, con-
by wetting with ethanol and washing three times for 5 min in sistent with the molecular mass of the monomeric protein. The
TTBS (50 mM Tris-HCl, pH 7.0, 137 mM NaCl, 2.7 mM KCl, relaxation data were analyzed according to the model-free
0.05% Tween 20). After overnight blocking at 4 °C in MBS (2% approach of Lipari and Szabo (42, 43) using TENSOR2 (44)
dry milk in TTBS), membranes were incubated for 1.5 h at 37 °C (supplemental Fig. S2).
with polyclonal antisera (1:3000 in MBS) followed by secondary Rigid Body Fitting—The procedure used to accommodate the
goat anti-mouse IgG alkaline phosphatase-conjugated antibod- NMR D1 structure into the EM map of the whole pilus previ-
ies (1:5000 in MBS; Promega), and signals were developed by ously generated (24) followed the same procedures used for the
using Western Blue Stabilized Substrate for Alkaline Phospha- fitting of the RrgBD2-D4 x-ray crystal structure and the D1
tase. For image processing, membranes were scanned using an computer model into the EM pilus map (22). A preliminary
Epson V750 Pro scanner at 800 dpi, 48-bit color depth and with rigid body fitting of the D2–D4 crystal fragment was performed
gamma 1.0 full linear response. by using CHIMERA (45– 47) followed by a rigid body fitting of
NMR Characterization of RrgB D1 Domain—Expression and the D1 NMR coordinates into the leftover N-terminal apical
purification of labeled D1 were carried out as described in the volume of the pilus. The D1 NMR coordinates were first fitted
supplemental Materials and Methods. NMR spectra were manually using CHIMERA by placing as much of the atomic
acquired at 298 K on Avance 900, 800, 700, and 500 MHz structure as possible into the EM density map, approximately in
Bruker spectrometers, all equipped with a triple resonance the position thought to be correct. This step was then followed
cryoprobe. The NMR experiments, used for the backbone and by a rigid-body fitting using the “fit model in map” tool from
the aliphatic side chain resonances assignment recorded on CHIMERA. This tool calculates, for the selected atoms, a posi-
13
C/15N and 15N enriched samples or on unlabeled D1 samples, tion that maximizes locally the sum of the densities. The eval-
are summarized in supplemental Table S3. Backbone dihedral uation of the correlation coefficient values, the resulting aver-
angle constraints were derived from 15N, 13C’, 13C␣, 13C␤, and age map value at the fit atoms, the number of fits atoms outside

14590 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 16 • APRIL 22, 2011
 Protective RrgB D1 Domain Involved in Pilus Polymerization

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

FIGURE 2. RrgB domains are protective in active immunization experiments. A and C, bacteremia. Circles represent the log cfu/ml of blood for individual
animals; horizontal bars represent the mean value of the log cfu/ml ⫾ S.E. for the group; the dotted line marks the detection limit (values under the dotted line
correspond to animals in which no cfu were detected). The treatment groups are indicated on the bottom. B and D, survival. The survival course for each group
is represented. The treatment groups are indicated close to each of the corresponding survival lines. A and B, n ⫽ 13 for D2 group, 15 for D4 and control (alum
ctrl) groups, 16 for the remaining groups. C and D, n ⫽ 31 for each of the groups. ***, p ⬍ 0.001; **, p ⬍ 0.01; *, p ⬍ 0.05.

the lowest value contour surface displayed were the parameters values were similar to those afforded by the FL RrgB and by the
used to assess the fit of the D1 molecule. combination of the four domains D1⫹D2⫹D3⫹D4. The
results of mortality are reported in Fig. 2B and supplemental
RESULTS Table S5A. D1 and D4 conferred significant increase of
Distinct Domains of RrgB Provide Protection in Active Immu- survival time, similar to FL RrgB and the combination
nization Experiments—The protective efficacy of each of the D1⫹D2⫹D3⫹D4. In particular, the median survival for the D1
four RrgB domains or of a mixture of them was assessed in a group was 2.5 days higher respect to that of the control group (4
mouse model of intraperitoneal challenge (TIGR4) in two dis- versus 1.5 days). At the end of the mortality observation, a sig-
tinct experiments, performed under the same conditions, nificant survival rate was found for D1 (44% survival), D4 (27%),
which were combined to reach n ⫽ 13–16 mice/group. The FL RrgB (44%), and D1⫹D2⫹D3⫹D4 (31%) groups.
results are summarized in Fig. 2, A and B, and detailed statistical N-terminal D1 Domain Is the Most Protective in Vivo—
analysis is provided in supplemental Table S5A. Among the single RrgB domains, D1 and D4 showed the most
All RrgB domains except D3 afforded significant protection significant protective efficacy and were therefore analyzed
against bacteremia (Fig. 2A), giving a reduction of the cfu geo- further in a larger group of mice. Four different experiments,
metric mean by 1–2 logs with respect to the controls. These carried out under the same conditions, were combined to

APRIL 22, 2011 • VOLUME 286 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 14591
Protective RrgB D1 Domain Involved in Pilus Polymerization
reach n ⫽ 31 animals/group. The results are shown in Fig. 2,
C and D, and detailed statistical analysis is provided in sup-
plemental Table S5B. In terms of bacteremia (Fig. 2C), both
D1 and D4 afforded highly significant protection, with a cfu
geometric mean by 2.6 and 1.5 logs lower, respectively, than
that of the control group, and 8 animals from the D1 group in
which cfu were undetectable. The reduction of bacteremia
was significantly superior in D1 than in the D4 group.
In terms of mortality course (Fig. 2D), both D1 and D4 con-
ferred significant protection. The increase of survival time
afforded by D1 showed a better trend than that of D4. In par-
ticular, only for the D1 group was the median survival time
higher than that of the control group (7.5 versus 1.5 days). At
the end of the mortality observation, the D1 group showed the
highest survival rate, i.e. 45% versus 21% observed for D4. An
evident difference of survival rates between D1 and D4 groups

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

was observed.
The possible relevance of antibodies in the protection elic-
ited by D1 and D4 was investigated by a passive serum transfer
experiment, with groups of 8 mice. The results are shown in Fig.
3, and detailed statistical analysis is provided in supplemental
Table S5C. Both anti-D1 and anti-D4 sera elicited significant
protection against bacteremia (Fig. 3A), with cfu geometric
means lower by 2.6 and 1.6 logs, respectively, than that of the
control group. Only anti-D1 serum afforded significant protec-
tion against mortality, giving 100% survival rate, whereas
santi-D4 gave a protective trend, with 60% survival rate. These
results indicate that immunization with D1 domain and at a
lower extent D4 domain, elicits functional antibodies that may
play a role in the protection.
D1 and D4 Antisera Recognize the Native Pilus and Linear
Epitopes within RrgB—To investigate the differences in protec-
tive efficacy exerted by the isolated RrgB domains with respect
to the FL RrgB in the in vivo assays, mouse sera were tested
for their capability of recognizing the native pilus and the
recombinant FL RrgB. Sera were probed against entire TIGR4
FIGURE 3. Anti-RrgB D1 and D4 sera are protective in passive serum trans-
bacteria by FACS analysis. Sera raised against D1, D4, and fer experiments. Symbols are described in the Fig. 2 legend. n ⫽ 8 for each of
D1⫹D2⫹D3⫹D4 gave a fluorescence intensity comparable the groups.
with that obtained with anti-FL RrgB, whereas D2- and D3-spe-
cific antisera recognized the native pilus less effectively. To detected a unique linear epitope (D4-1) spanning residues 494 –
explain the lower recognition efficacy observed with D2 and 508 (Fig. 4C).
D3, the same sera were titrated in ELISA against the recombi- Solution Structure of the D1 Domain—The solution structure
nant FL RrgB. As shown in supplemental Fig. S3, antibody titers of D1 was investigated by NMR spectroscopy. Its 1H-15N HSQC
elicited by D2 and D3 immunization toward FL RrgB were spectrum showed well dispersed resonances indicative of an
about 10 times lower than those obtained by immunization overall well folded protein (supplemental Fig. S4). D1 showed a
with D1, D4, D1⫹D2⫹D3⫹D4, and FL RrgB, consistent with common IgG-like ␤ sandwich fold (41 Å ⫻ 48 Å ⫻ 30 Å) and a
the results obtained by FACS analysis on the polymerized topology of secondary structure elements drawn in Fig. 5. The
native RrgB. core of the structure was formed by seven parallel and antiparallel
To gain more insights on the epitopes recognized by the pro- ␤-strands: ␤1(36–39), ␤4(80–85), ␤7(119–121), ␤8(127–130),
tective D1 and D4 polyclonal antibodies, a PepScan approach, ␤9(138–143), ␤10(166 –169), and ␤11(178 –180). These ␤-strands
suitable for identifying linear epitopes, was applied. Overlap- were arranged in two sheets (comprising ␤1, ␤8, ␤11 and ␤4, ␤7, ␤9,
ping 15-mer peptides with an offset of 5 residues were synthe- ␤10, respectively) packed against each other and flanked by two
sized in situ on a glass fiber membrane. The library of peptides long segments (40 –78, 87–117) located between strands ␤1 and ␤4
tested covered residues 25–190 of D1 and 444 – 628 of D4. and strands ␤4 and ␤7, respectively. An ␣-helix (49–57), flanked by
Incubation with the anti-D1 serum (previously used in passive two short ␤-strands ␤2 (42–44) and ␤3 (73–75), was inserted
protection experiments) revealed a single linear epitope cover- within the first segment. Two additional ␤-strands ␤5 (89–91) and
ing residues 40 –59 (D1-1) (Fig. 4B), whereas anti-D4 serum ␤6 (97–101) formed a ␤-hairpin structure inserted within the sec-

14592 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 16 • APRIL 22, 2011
 Protective RrgB D1 Domain Involved in Pilus Polymerization

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

FIGURE 4. Polyclonal antibodies raised against D1 and D4 recognize the native pilus and linear epitopes within RrgB efficiently. A, TIGR4 bacteria were
incubated with mouse primary antibodies directed against the specified recombinant proteins (1:400 dilution) followed by FITC-conjugated goat anti-mouse IgG
(1:100 dilution). Bacterial staining was analyzed by flow cytometry (FACS-Calibur). Mouse control sera (immunized with PBS plus alum) were used as negative control.
B and C, glass fiber membranes with arrayed peptides synthesized in situ covering residues 25–190 (D1) and 444 – 628 (D4) of RrgB were incubated with anti-D1 (A) or
anti-D4 (B) polyclonal mouse antibodies (1:3000) and then with goat anti-mouse IgG alkaline phosphatase-conjugated antibodies (1:5000). Linear epitopes corre-
sponding to peptide sequences recognized by the antibodies are reported. Underlining marks common residues present in adjacent peptides in the PepScan.

The structure was well defined with the exception of three

long loops corresponding to the stretches 56 – 69, 148 –162,
and 173–177 (supplemental Fig. S1). Heteronuclear relaxation
measurements revealed that residues in the first two loops had
heteronuclear NOE and longitudinal R1 values lower and
higher than average, respectively (supplemental Fig. S2). This
behavior was a consequence of local internal motions occurring
on a faster time scale with respect to the overall reorientational
correlation time (␶r) of the molecule and accordingly, a corre-
lation time (␶e) for these fast motions can be fitted for the pre-
vious mentioned loops (supplemental Fig. S2). In addition,
conformational exchange processes, occurring on the millisec-
ond-microsecond scale, affected some residues located in the
region 155–164, as monitored by transverse R2 relaxation rates
higher than the average (supplemental Fig. S2). These data indi-
cated that these loops experience higher flexibility, showing
accordingly a low number of long ranges 1H-1H NOEs (supple-
mental Fig. S5). For a few residues, the assignment of the back-
bone resonances was also not achieved (Glu-143, His-145, Ser-
146, Ser-148, Thr-149, Tyr-150, Val-152, and Gly-160), likely as
a consequence of an increased local mobility. The loop between
strands ␤9 and ␤10 (residues 148 –162) was the most disordered,
with a r.m.s.d. value of about 2 Å (supplemental Fig. S1). Con-
formational exchange processes on the millisecond-microsec-
ond time scale are observed also for the loop between strands
␤10 and ␤11 comprising residues 174 –178.
FIGURE 5. Solution structure of the D1 domain. A, ribbon diagram with the The protein core is characterized by hydrophobic interac-
secondary structure elements. ␤-Strands are shown in cyan, the ␣-helix in red. tions between residues located on the first (strands ␤1, ␤8, ␤11)
B, topology diagram. The ␣-helix is represented by a red cylinder, and the
␤-strands are cyan arrows. and second (strands ␤4, ␤7, ␤9, ␤10) sheets. A salt bridge
between two complementarily charged side chains of residues
ond segment. In 50% of 20 conformers of the D1 family an addi- Lys-41 and Glu-143 was also present. The aliphatic side chains
tional ␤-sheet was formed by two short hydrogen-bonded of residues Met-48, Ile-52, Ala-53, and Leu-56, all located on
␤-strands (stretches 161–163 and 184–187). one side of the ␣-helix, formed hydrophobic interactions with

APRIL 22, 2011 • VOLUME 286 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 14593
Protective RrgB D1 Domain Involved in Pilus Polymerization
mapped onto the FL model of RrgB, confirmed their superficial
localization (Fig. 6B).
Lysine 183 of D1 Is Required for Intermolecular Isopeptide
Bond Formation and Pilus Polymerization—Pili of Gram-posi-
tive bacteria are polymerized by means of intermolecular iso-
peptide bonds occurring between the Thr of the C-terminal
LPXTG motif of a RrgB molecule and a Lys located at the N
terminus of the following molecule (18, 29, 30). To identify the
Lys implicated in the intermolecular isopeptide bond forma-
tion between two consecutive RrgB monomers, a TIGR4 RrgB
deletion mutant (no longer able to assemble a pilus on its sur-
face) was created. Subsequently, RrgB expression and pilus
polymerization were restored in TIGR4⌬RrgB by transforming
the mutant with plasmids expressing either wild-type RrgB or
FIGURE 6. Analysis of RrgB linker flexibility through superimposition and
modeling. A, superimposition of the D1 domain (blue) and the C. diphtheriae RrgB mutated in single Lys residues (Lys 3 Ala substitutions).
SpaA N-terminal domain (red). The position of Lys-190 residue, forming the Sequence analysis revealed that the D1 contains the canonical

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

intermolecular isopeptide bond between two neighboring SpaA molecules, 181
YPKN184 pilin motif, with Lys-183 nicely superimposing
is shown along with residue Lys-183 of RrgB, which occupies a similar posi-
tion, and is indicated in a black circle. The missing loop in the SpaA crystal onto the functional SpaA Lys-190, as shown in Fig. 6A.
structure is indicated as a gray dotted line. B, modeled conformation of the Noteworthy, D1 also presents the sequence fragment
full-length RrgB molecule obtained by combining the D1 NMR coordinates 160
with the RrgB D2-D4 x-ray coordinates. D1-1 and D4-1 epitopes (residues GSKAVP165, similar to the motif containing the Lys residue
40 –59 and 494 –508 respectively) are rendered as yellow spheres. functional for the pilus assembly in S. pyogenes Spy0128 (31).
Lys-183 and Lys-162, along with two additional Lys residues
(Lys-138 and Lys-309), located on D1 and D3 domains, respec-
aliphatic residues of ␤2 and ␤3 strands; these interactions deter- tively, were selected and mutated.
mined the position of the helix with respect to the rest of the WB analysis performed on whole cell bacterial lysates using
protein. rabbit polyclonal antisera raised against RrgA, RrgB, or RrgC
A search for related protein structures performed through revealed that all of the pilus proteins were expressed in all of the
the DALI Server (48) retrieved the N-terminal domain of the complemented mutants (Fig. 5). However, the typical pilus-as-
SpaA pilus backbone protein of C. diphtheriae (Protein Data sociated high molecular weight ladder was revealed only in the
Bank (PDB) code 3HTL; r.m.s.d. 2.1 Å), the C-terminal CNA3 case of TIGR4⌬RrgB complemented with RrgB wild type or
domain of the major pilin protein of Bacillus cereus BcpA (PDB RrgB mutated in Lys-162, Lys-138, or Lys-309. In contrast, in
code 3KPT; r.m.s.d. 2.4 Å), and the N1 domain of the Strepto- the mutant complemented with K183A, RrgB was detectable
coccus agalactiae minor pilin GBS52 (PDB code 2PZ4; r.m.s.d. only as a monomer (Fig. 7), clearly indicating that Lys-183
3.4 Å). Like D1, none of these domains contained intramolecu- is the residue implicated in intermolecular isopeptide bond
lar isopeptide bonds. However, only the SpaA domain is located formation.
at the N terminus of pilus backbone protein as D1; their overlay Moreover, in the presence of the RrgB K183A substitution
is presented in Fig. 6A. only hetero-oligomers composed of RrgA-RrgC (about 160
In an attempt to determine the orientation of D1 with respect kDa) or RrgB-RrgC (about 110 kDa) could be detected by WB
to the D2–D4 RrgB portion, a rigid body fitting of all the analysis (see arrows in Fig. 7, A and C). The absence of RrgA-
domains into the shape of the native pilus obtained from cryo- RrgB heterodimers suggests that RrgA and RrgB are linked
electron microscopy (cryo-EM) was attempted (24). Initial rigid exclusively through intermolecular isopeptide bonds involving
body fitting of the D2–D4 crystal structure into the cryo-EM the D1 residue Lys-183 of RrgB.
density map of the native pilus left an apical unoccupied vol-
ume, likely due to the absence of D1. However, when the simul- DISCUSSION
taneous fitting of the D1 and the D2–D4 domains was carried Ever since their initial discovery, pili of Gram-positive bacte-
out, some portions of D1 could not be accommodated into the ria have received considerable attention because they are asso-
apical empty volume (supplemental Fig. S6 and supplemental ciated with a number of different virulence mechanisms (15, 16,
Table S6). To model the FL RrgB, we merged the D1 and 33) and elicit protection in animal models (32, 49 –52). In par-
D2–D4 structures into a single molecule by overlapping resi- ticular, the S. pneumoniae pilus was found to be implicated in
dues 188 –191, shared by the C terminus of D1 and the N ter- the initial attachment to epithelial cells (15, 16, 33), and the
minus of D2–D4. The relative orientation of D1 with respect to pilus components, being protective in mouse models of infec-
the others domains was then varied to best fit into the cryo-EM tion, are regarded as potential vaccine candidates (32, 33). The
map (Fig. 6B). The final model, which represents only one of the major pilin RrgB is organized into four Ig-like domains (D1–
possible orientations of D1 with respect to the rest of the pro- D4), as shown by combined structural approaches (Ref. 24 and
tein, does not present steric clashes between D1 and the D2–D4 this work). We have tested the four RrgB domains in animal
domains. The two linear epitopes, previously identified to be model experiments and have shown that the protective efficacy
located within the D1 and D4 domains by peptide hybridization exerted by the combination of the four RrgB domains
with specific protective polyclonal antibodies (Fig. 4), when D1⫹D2⫹D3⫹D4 is comparable with that afforded by the FL

14594 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 16 • APRIL 22, 2011
 Protective RrgB D1 Domain Involved in Pilus Polymerization

FIGURE 7. Lys-183 of RrgB is implicated in intermolecular isopeptide bond formation. WB analysis was performed using polyclonal rabbit antisera against
RrgA (A), RrgB (B), and RrgC (C). In all panels lanes are loaded as follows: 1, T4 WT; 2, T4⌬RrgB; 3, T4⌬RrgBRrgBWT; 4, T4⌬RrgBRrgB(K138A); 5,
T4⌬RrgBRrgB(K162A); 6, T4⌬RrgBRrgB(K183A); 7, T4⌬RrgBRrgB(K309A); 8, T4⌬RrgBpMU1328; 9, molecular mass marker. *, RrgA monomer (estimated
molecular mass of native monomeric RrgA is 92 kDa). **, RrgB monomer (estimated molecular mass of native monomeric RrgB is 65 kDa). Arrows, the band

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

migrating at an apparent molecular mass of 160 kDa indicated by an arrow in lane 2 and 6 of A, and C is compatible with an RrgA⫹RrgC complex; the band at
110 kDa present in lane 6 of B and C could correspond to an RrgC⫹RrgB complex.

RrgB. Among the single domains, D1 is the most protective, and tein structural plasticity could be related to the ability of D1 to
D4 retained an important part of the protective efficacy of the undergo local conformational changes and to adapt its struc-
FL protein. The lower protection achieved by D2 and D3 com- ture to optimize the interactions with the antibodies and
pared with the FL protein, as well as with the D1 and D4, is increase the affinity and the specificity of the antigen-antibody
probably because the antibodies elicited by the former two recognition process. The dynamics of D1 could therefore
domains recognize the FL protein less efficiently, in both its strongly contribute to the interface adaptation for molecular
native and recombinant forms. recognition such that the antibody can select an optimal con-
This may be the result of smaller exposed surface areas expe- former from a wide distribution of possible D1 conformations.
rienced by D2 and D3 in the FL RrgB compared with the D2 and The rigid structure of the D2–D4 region prevents such an effec-
D3 isolated domains and with D1 and D4. It is possible, in fact, tive conformational selection for these domains. The above
that the antibodies generated against the isolated domains are described phenomenon has been observed for other protein-
recognizing areas of D2 and D3 that are buried in the FL pro- protein or protein-DNA interaction processes (53, 54).
tein. Alternatively, D2 and D3 could assume a slightly different To shed light on the molecular mechanism driving pilus
conformation when expressed as single domains, thus generat- polymerization in S. pneumoniae, we investigated which of the
ing nonfunctional antibodies. On the other hand, the two linear lysine residues of D1 was engaged in the intermolecular isopep-
epitopes (Fig. 6B) identified within D1 and D4 by PepScan anal- tide bond formation. Site-directed mutagenesis followed by
ysis performed with protective polyclonal antibodies raised complementation identified Lys-183 as crucial for the pilus
against the two domains (conformational epitopes are not assembly. This result is consistent with the observation that the
detectable with this method) are well exposed on the surface of spatial position of RrgB Lys-183 can be superimposed onto Lys-
the RrgB molecule and could contribute to the protective activ- 190 of the C. diphtheriae pilus backbone subunit SpaA, known
ity exerted by D1 and D4. Further experiments are needed to to be involved in the intermolecular isopeptide linkage (30).
understand to what extent these linear epitopes contribute to Interestingly, as shown in Fig. 6A, both lysines are not fully
the protective activity exerted by the two domains. Taken available to form an external bond. In particular, the average
together, these results suggest that RrgB contains multiple pro- relative solvent accessibility of the Lys-183 over the D1 family of
tective epitopes, thus confirming the potential of this vaccine conformers is 27.2% ⫾ 4.8, as the Lys side chain projects into a
candidate. Furthermore, although the existence of possible cleft between the main body of the protein and the segment
conformational epitopes involving residues from different 40 –78 containing the mobile loop 56 – 69 (27). This suggests
domains cannot be excluded, their contribution to the overall that pilus backbone proteins, to be polymerized, might undergo
protective efficacy might not be essential. conformational changes, probably involving not only the Lys
To obtain more information about the structural role of D1 residue (Lys-183) but also the flexible regions spatially close to
and to try to correlate it with the protection data discussed it (49 – 69, 152–167, and 183–193), to allow the formation of
above, we solved the solution structure of this domain by using the covalent intermolecular isopeptide bond. NMR mobility
NMR spectroscopy. D1 shows an Ig-like fold, does not contain data indicate that the C terminus of the D1, where Lys-183 is
any intramolecular isopeptide bond, and has many flexible located, and other loops are highly mobile and that such
regions. The observed D1 flexibility, indeed, could play a fun- dynamics could be relevant for the intermolecular isopeptide
damental role in the specific antigen-antibody recognition bond formation (supplemental Fig. S2). Consistently, the
process (53), thus accounting for D1 enhanced protection capa- absence of stabilizing intramolecular isopeptide bonds renders
bility with respect to the more rigid D2–D4 domains, each one D1, unlike the other domains, less rigid and prone to conforma-
containing an intramolecular isopeptide bond. In fact, the pro- tional rearrangements. Furthermore, the occurrence of a struc-

APRIL 22, 2011 • VOLUME 286 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 14595
Protective RrgB D1 Domain Involved in Pilus Polymerization
tural rearrangement of D1 within the native pilus structure is 8. Melegaro, A., Choi, Y. H., George, R., Edmunds, W. J., Miller, E., and Gay,
also in line with the partial fitting of its NMR structure onto the N. J. (2010) BMC Infect. Dis. 10, 90
9. Millar, E. V., O’Brien, K. L., Zell, E. R., Bronsdon, M. A., Reid, R., and
molecular shape of the native pilus determined by cryo-EM.
Santosham, M. (2009) Pediatr. Infect. Dis. J. 28, 711–716
Mutagenesis and complementation data were used to ana- 10. Barocchi, M. A., Ries, J., Zogaj, X., Hemsley, C., Albiger, B., Kanth, A.,
lyze the covalent links established among the three pilus pro- Dahlberg, S., Fernebro, J., Moschioni, M., Masignani, V., Hultenby, K.,
teins either in the absence of RrgB or in the presence of the RrgB Taddei, A. R., Beiter, K., Wartha, F., von Euler, A., Covacci, A., Holden,
K183A mutant, in an attempt to provide further insights into D. W., Normark, S., Rappuoli, R., and Henriques-Normark, B. (2006) Proc.
the possible organization of the native pilus. In the presence of Natl. Acad. Sci. U.S.A. 103, 2857–2862
11. Dramsi, S., Caliot, E., Bonne, I., Guadagnini, S., Prévost, M. C., Kojadi-
the nonpolymerizing RrgB K183A mutant, the lack of RrgA-
novic, M., Lalioui, L., Poyart, C., and Trieu-Cuot, P. (2006) Mol. Microbiol.
RrgB heterodimers provides evidence that RrgA and RrgB are 60, 1401–1413
unidirectionally linked only through the Lys-183 of RrgB and 12. Lauer, P., Rinaudo, C. D., Soriani, M., Margarit, I., Maione, D., Rosini, R.,
the C-terminal Thr of RrgA. This arrangement is in accordance Taddei, A. R., Mora, M., Rappuoli, R., Grandi, G., and Telford, J. L. (2005)
with the model proposed by Hilleringmann et al., positioning Science 309, 105
RrgA at the pilus terminus, thus ruling out the alternative pos- 13. LeMieux, J., Hava, D. L., Basset, A., and Camilli, A. (2006) Infect. Immun.
74, 2453–2456
sibility of RrgA being incorporated along the pilus shaft (22). 14. Mora, M., Bensi, G., Capo, S., Falugi, F., Zingaretti, C., Manetti, A. G.,
Conversely, an RrgA-RrgC multimer is detectable either when Maggi, T., Taddei, A. R., Grandi, G., and Telford, J. L. (2005) Proc. Natl.

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

RrgB is not expressed, as already reported by Falker et al. (20) Acad. Sci. U.S.A. 102, 15641–15646
and Le Mieux et al. (55), or in the presence of nonpolymerizing 15. Hilleringmann, M., Giusti, F., Baudner, B. C., Masignani, V., Covacci, A.,
RrgB K183A. In this case, the mutated RrgB is competing with Rappuoli, R., Barocchi, M. A., and Ferlenghi, I. (2008) PLoS Pathog. 4,
e1000026
RrgA for the linkage to RrgC, as demonstrated by the presence
16. Nelson, A. L., Ries, J., Bagnoli, F., Dahlberg, S., Fälker, S., Rounioja, S.,
of RrgB-RrgC hetero-oligomers (Fig. 7, B and C). Concomitant Tschöp, J., Morfeldt, E., Ferlenghi, I., Hilleringmann, M., Holden, D. W.,
detection of RrgA-RrgC hetero-oligomers even under these Rappuoli, R., Normark, S., Barocchi, M. A., and Henriques-Normark, B.
conditions further strengthens the idea that in wild-type bacte- (2007) Mol. Microbiol. 66, 329 –340
ria, although not detectable by electron microscopy analysis of 17. Telford, J. L., Barocchi, M. A., Margarit, I., Rappuoli, R., and Grandi, G.
purified pili, a fraction of RrgA and RrgC might be directly (2006) Nat. Rev. Microbiol. 4, 509 –519
18. Ton-That, H., and Schneewind, O. (2004) Trends Microbiol. 12, 228 –234
linked to each other.
19. El Mortaji, L., Terrasse, R., Dessen, A., Vernet, T., and Di Guilmi, A. M.
In conclusion, this study provides additional information (2010) J. Biol. Chem. 285, 12405–12415
elucidating pilus proteins features and also paves the way to the 20. Fälker, S., Nelson, A. L., Morfeldt, E., Jonas, K., Hultenby, K., Ries, J.,
rational design of new RrgB-based molecules to implement a Melefors, O., Normark, S., and Henriques-Normark, B. (2008) Mol. Mi-
protein-based vaccine against pneumococcal disease. More- crobiol. 70, 595– 607
over, the newly acquired structural and dynamic information 21. Manzano, C., Contreras-Martel, C., El Mortaji, L., Izoré, T., Fenel, D.,
Vernet, T., Schoehn, G., Di Guilmi, A. M., and Dessen, A. (2008) Structure
on the RrgB molecule provided by this study suggests that the
16, 1838 –1848
conformational flexibility of D1 is pivotal for the protein-anti- 22. Hilleringmann, M., Ringler, P., Müller, S. A., De Angelis, G., Rappuoli, R.,
body recognition process. These findings together with the new Ferlenghi, I., and Engel, A. (2009) EMBO J. 28, 3921–3930
functional information could be used to better understand pilus 23. Izoré, T., Contreras-Martel, C., El Mortaji, L., Manzano, C., Terrasse, R.,
functions and its role in pathogenesis. Vernet, T., Di Guilmi, A. M., and Dessen, A. (2010) Structure 18, 106 –115
24. Spraggon, G., Koesema, E., Scarselli, M., Malito, E., Biagini, M., Norais, N.,
Emolo, C., Barocchi, M. A., Giusti, F., Hilleringmann, M., Rappuoli, R.,
Acknowledgments—We thank Giacomo Matteucci and Tommaso Lesley, S., Covacci, A., Masignani, V., and Ferlenghi, I. (2010) PLoS One 5,
Pasquali, who managed Animal Resources; Marco Tortoli, Stefania e10919
Torricelli, Luigi Manganelli, and Elena Amantini, who took care of 25. Budzik, J. M., Poor, C. B., Faull, K. F., Whitelegge, J. P., He, C., and Sch-
the animal treatments; Silvia Maccari, Esmeralda Bizzarri, and Ales- neewind, O. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 19992–19997
sia Corrado, who lent technical assistance for the in vivo experiments; 26. Kang, H. J., and Baker, E. N. (2009) J. Biol. Chem. 284, 20729 –20737
and Morena Lo Sapio for technical assistance in FACS and ELISA 27. Kang, H. J., Paterson, N. G., Gaspar, A. H., Ton-That, H., and Baker, E. N.
experiments. Intercell AG kindly provided us with pMU1328. Luisa (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16967–16971
Lozzi and Luisa Bracci (University of Siena) synthesized the peptides 28. Alegre-Cebollada, J., Badilla, C. L., and Fernández, J. M. (2010) J. Biol.
onto the glass fiber membranes used for Pepscan analysis. Finally, we Chem. 285, 11235–11242
29. Guttilla, I. K., Gaspar, A. H., Swierczynski, A., Swaminathan, A., Dwivedi,
thank Mariagrazia Pizza for a critical review of the manuscript.
P., Das, A., and Ton-That, H. (2009) J. Bacteriol. 191, 5603–5612
30. Ton-That, H., and Schneewind, O. (2003) Mol. Microbiol. 50, 1429 –1438
31. Kang, H. J., Coulibaly, F., Clow, F., Proft, T., and Baker, E. N. (2007) Science
REFERENCES 318, 1625–1628
1. Fletcher, M. A., and Fritzell, B. (2007) Vaccine 25, 2507–2512 32. Gianfaldoni, C., Censini, S., Hilleringmann, M., Moschioni, M., Facciotti,
2. Kim, K. S. (2010) Lancet Infect. Dis. 10, 32– 42 C., Pansegrau, W., Masignani, V., Covacci, A., Rappuoli, R., Barocchi,
3. O’Brien, K. L., Wolfson, L. J., Watt, J. P., Henkle, E., Deloria-Knoll, M., M. A., and Ruggiero, P. (2007) Infect. Immun. 75, 1059 –1062
McCall, N., Lee, E., Mulholland, K., Levine, O. S., and Cherian, T. (2009) 33. Moschioni, M., Emolo, C., Biagini, M., Maccari, S., Pansegrau, W., Donati,
Lancet 374, 893–902 C., Hilleringmann, M., Ferlenghi, I., Ruggiero, P., Sinisi, A., Pizza, M.,
4. Pelton, S. I., and Leibovitz, E. (2009) Pediatr. Infect. Dis. J. 28, S133–137 Norais, N., Barocchi, M. A., and Masignani, V. (2010) Infect. Immun. 78,
5. Ryan, M. W., and Antonelli, P. J. (2000) Laryngoscope 110, 961–964 5033–5042
6. van der Poll, T., and Opal, S. M. (2009) Lancet 374, 1543–1556 34. Achen, M. G., Davidson, B. E., and Hillier, A. J. (1986) Gene 45, 45– 49
7. Melegaro, A., Gay, N. J., and Medley, G. F. (2004) Epidemiol. Infect. 132, 35. Alloing, G., Martin, B., Granadel, C., and Claverys, J. P. (1998) Mol. Mi-
433– 441 crobiol. 29, 75– 83

14596 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 16 • APRIL 22, 2011
 Protective RrgB D1 Domain Involved in Pilus Polymerization
36. Cornilescu, G., Delaglio, F., and Bax, A. (1999) J. Biomol. NMR 13, (2006) BMC Bioinformatics 7, 339
289 –302 47. Goddard, T. D., Huang, C. C., and Ferrin, T. E. (2005) Structure 13,
37. Herrmann, T., Güntert, P., and Wüthrich, K. (2002) J. Mol. Biol. 319, 473– 482
209 –227 48. Holm, L., and Sander, C. (1996) Methods Enzymol. 266, 653– 662
38. Pearlman, A. D., Case, D. A., Caldwell, J. W., Ross, W. S., Cheatman, T. E., 49. Abbot, E. L., Smith, W. D., Siou, G. P., Chiriboga, C., Smith, R. J., Wilson,
III, DeBolt, S., Ferguson, D., Seibel, G., and Kollman, P. (1995) Comp. Phys. J. A., Hirst, B. H., and Kehoe, M. A. (2007) Cell. Microbiol. 9, 1822–1833
Commun. 91, 1– 41 50. Pezzicoli, A., Santi, I., Lauer, P., Rosini, R., Rinaudo, D., Grandi, G., Tel-
39. Koradi, R., Billeter, M., and Wuthrich, K. (1996) J. Mol. Graph. 14, ford, J. L., and Soriani, M. (2008) J. Infect. Dis. 198, 890 – 898
29 –32 51. Rosini, R., Rinaudo, C. D., Soriani, M., Lauer, P., Mora, M., Maione, D.,
40. Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Taddei, A., Santi, I., Ghezzo, C., Brettoni, C., Buccato, S., Margarit, I.,
Gish, G., Shoelson, S. E., Pawson, T., Forman-Kay, J. D., and Kay, L. E. Grandi, G., and Telford, J. L. (2006) Mol. Microbiol. 61, 126 –141
(1994) Biochemistry 33, 5984 – 6003 52. Margarit, I., Rinaudo, C. D., Galeotti, C. L., Maione, D., Ghezzo, C., But-
41. Grzesiek, S., and Bax, A. (1993) J. Am. Chem. Soc. 115, 12593–12594 tazzoni, E., Rosini, R., Runci, Y., Mora, M., Buccato, S., Pagani, M., Tre-
42. Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546 – 4559 soldi, E., Berardi, A., Creti, R., Baker, C. J., Telford, J. L., and Grandi, G.
43. Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4559 – 4570 (2009) J. Infect. Dis. 199, 108 –115
44. Dosset, P., Hus, J. C., Blackledge, M., and Marion, D. (2000) J. Biomol. 53. Mittag, T., Kay, L. E., and Forman-Kay, J. D. (2010) J. Mol. Recognit. 23,
NMR 16, 23–28 105–116
45. Goddard, T. D., Huang, C. C., and Ferrin, T. E. (2007) J. Struct. Biol. 157, 54. Dyson, H. J., and Wright, P. E. (2005) Nat. Rev. Mol. Cell Biol. 6,
281–287 197–208

Downloaded from www.jbc.org at UNIVERSITA DI FIRENZE, on November 3, 2011

46. Meng, E. C., Pettersen, E. F., Couch, G. S., Huang, C. C., and Ferrin, T. E. 55. LeMieux, J., Woody, S., and Camilli, A. (2008) J. Bacteriol. 190, 6002– 6013

APRIL 22, 2011 • VOLUME 286 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 14597
View publication stats