Montanari et al_Cellular_Microbiology_2011

Human heat shock protein (Hsp) 90 interferes with
Neisseria meningitidis adhesin A (NadA)-mediated
adhesion and invasion
Paolo Montanari,1†
Giuseppe Bozza,1
Barbara Capecchi,1
Elena Caproni,1
Riccardo Barrile,1
Nathalie Norais,1
Mirco Capitani,2
Michele Sallese,2
Paola Cecchini,3,4‡
Laura Ciucchi,1
Zhenai Gao,5
Rino Rappuoli,1
Mariagrazia Pizza,1
Beatrice Aricò1
and Marcello Merola1,6
*
1
Research Center, Novartis Vaccines and Diagnostics,
Via Fiorentina 1, 53100 Siena, Italy.
2
Unit of Genomic Approaches to Membrane Traffic,
Consorzio Mario Negri Sud, Via Nazionale 8/A, 66030
S. Maria Imbaro (CH), Italy.
3
Dipartimento di Scienze Biomediche Sperimentali,
Università di Padova, Via U. Bassi 58/B, I-35131
Padova, Italy.
4
Centro di Ricerca Interdipartimentale per le
Biotecnologie Innovative, Università di Padova, Via U.
Bassi 58/B, I-35131 Padova, Italy.
5
Novartis Institute for Biomedical Research, 4560
Horton St. M/S:4.5, Emeryville, CA 94608-2916, USA.
6
Dipartimento Biologia Strutturale e Funzionale,
Università di Napoli ‘Federico II’, Via Cinthia 21, 80126
Napoli, Italy.
Summary
NadA (Neisseria meningitidis adhesin A), a menin-
gococcal surface protein, mediates adhesion to
and invasion of human cells, an activity in which
host membrane proteins have been implicated.
While investigating these host factors in human
epithelial cells by affinity chromatography, we dis-
covered an unanticipated interaction of NadA with
heat shock protein (Hsp) 90, a molecular chaper-
one. The specific in vitro interaction of recombi-
nant soluble NadA and Hsp90 was confirmed by
co-immunoprecipitations, dot and far-Western
blot. Intriguingly, ADP, but not ATP, was required
for this association, and the Hsp90 inhibitor
17-AAG promoted complex formation. Hsp90
binding to an Escherichia coli strain used as
carrier to express surface exposed NadA con-
firmed these results in live bacteria. We also
examined RNA interference, plasmid-driven over-
expression, addition of exogenous rHsp90 and
17-AAG inhibition in human epithelial cells to
further elucidate the involvement of Hsp90 in
NadA-mediated adhesion and invasion. Together,
these data suggest an inverse correlation between
the amount of host Hsp90 and the NadA adhesive/
invasive phenotype. Confocal microscopy also
demonstrated that meningococci interact with cel-
lular Hsp90, a completely novel finding. Altogether
our results show that variation of host Hsp90
expression or activity interferes with adhesive and
invasive events driven by NadA.
Introduction
Neisseria meningitidis causes mortality worldwide due to
septicaemia and meningitis. Host–bacterial interaction for
this organism begins with epithelial colonization, followed
by invasion, intracellular persistence and transcytosis.
Although some meningococcal vaccines exist against
common pathogenic serogroups A, C, W-135 and Y, the
search for a broadly protective vaccine against serogroup
B required new means of antigen identification that
resulted in the discovery of surface-expressed proteins,
including potential new virulence factors. The character-
ization of these proteins is hoped to provide a better
understanding of meningococcal pathogenesis (Pizza
et al., 2000; Giuliani et al., 2006).
One such protein is NadA (Neisseria meningitidis
adhesin A), a phase-variable meningococcal surface-
exposed protein, present in three of the four known hyper-
virulent serogroup B lineages (Comanducci et al., 2002;
2004; Metruccio et al., 2009). NadA was included as a
major antigen in the multicomponent vaccine 4CMenB
(Giuliani et al., 2006; Bambini et al., 2009). NadA belongs
to the ‘Oca’ (Oligomeric coiled-coil adhesin) family, a sub-
group of the trimeric autotransporter adhesins (Surana
Received 15 April, 2011; revised 14 October, 2011; accepted 20
October, 2011. *For correspondence. E-mail marcello.merola@
novartis.com; Tel. (+39) 0577 243864; Fax (+39) 0577 243564.
Present addresses: †
Max Planck Institute for Infection Biology,
Charitéplatz 1, D-10117 Berlin, Germany; ‡
Babraham Research
Campus, Babraham, Cambridge CB22 3AT, UK.
Cellular Microbiology (2012) 14(3), 368–385 doi:10.1111/j.1462-5822.2011.01722.x
First published online 8 December 2011
© 2011 Blackwell Publishing Ltd
cellular microbiology

et al., 2004; Cotter et al., 2006; Linke et al., 2006). Gen-
erally, Oca proteins mediate bacterial interaction with
host cells or extracellular matrix (ECM) proteins or
induce invasion into target cells (Yang and Isberg,
1993; McMichael et al., 1998; Eitel and Dersch, 2002;
Laarmann et al., 2002; Ray et al., 2002; Roggenkamp
et al., 2003; Li et al., 2004; Riess et al., 2004; Zhang
et al., 2004; Girard and Mourez, 2006; Heise and Dersch,
2006; Scarselli et al., 2006; Serruto et al., 2009). Like
other Oca proteins, such as YadA of Yersinia spp. (Bliska
et al., 1993; Iriarte and Cornelis, 1996; El Tahir and
Skurnik, 2001), UspAs proteins of Moraxella catarrhalis
(Lafontaine et al., 2000; Hill and Virji, 2003), Vomp pro-
teins of Bartonella quintana (Zhang et al., 2004), BadA of
B. henselae (Riess et al., 2004) and HadA of Haemophi-
lus influenzae biogroup aegyptius (Serruto et al., 2009),
NadA has a conserved C-terminal membrane anchor
through which the protein is translocated to the cell
surface, a central alpha helical domain (stalk) with high
propensity to form coiled-coil structures, and an
N-terminal globular head that has been associated with
binding to specific cellular receptors.
NadA forms stable trimers on the bacterial surface and
thereby contributes to mediate N. meningitidis adhesion
to and invasion of epithelial cells. A trimeric protein and
properly folded N-terminal domain are necessary to
NadA-cell binding to human cells (Capecchi et al., 2005;
Tavano et al., 2011). A protein receptor molecule, which
is differentially expressed by various human epithelial
cell lines, appears to mediate the binding of the trimeric
NadA (Capecchi et al., 2005). The expression of full-
length NadA on the surface of Escherichia coli and
the purification of soluble recombinant NadAD351–405
(rNadA) have been described (Comanducci et al., 2002;
Capecchi et al., 2005). This soluble rNadA was included
in 4CMenB (Giuliani et al., 2006) and was shown to
induce high levels of bactericidal antibodies in various
models (Comanducci et al., 2002; Bowe et al., 2004;
Ciabattini et al., 2008). Further, rNadA activates human
monocyte-derived dendritic cells and monocytes/
macrophages (Mazzon et al., 2007; Franzoso et al.,
2008) and is recognized by convalescent serum from
children (Litt et al., 2004). These studies suggest that
rNadA retains the functional features of native NadA,
which is expressed and immunogenic in vivo.
As N. meningitidis has diverse, and likely redundant,
virulence factors (Virji, 2009) identifying the precise role of
NadA is difficult; however, E. coli expressing NadA helps
isolate its specific contribution. Surface-exposed NadA
behaves similarly in E. coli and meningococci, first
forming a stable trimer on the bacterial surface and then
mediating adhesion to and invasion of host cells. The host
cell mechanisms behind these activities remain some-
what poorly understood. To explore meningococcal infec-
tion on the molecular level, we used transformed E. coli
expressing surface NadA, rNadA and unencapsulated
N. meningitidis strains, to investigate the interaction of
NadA with epithelial cells. We discovered a novel interac-
tion between NadA and human Hsp90 that was promoted
by ADP and the specific Hsp90 inhibitor 17-AAG (17-N-
allylamino-17-demethoxygeldanamycin). Unexpectedly,
bacterial adhesion to and invasion of a human epithelial
cell line were inversely correlated with the expression of
Hsp90 by host cells.
Hsp90 cycles between an apo conformation, in the
absence of associated nucleotides, and two conforma-
tionally distinct types that are associated with ADP or ATP
(Mayer, 2010). Hsp90 primarily forms complexes in the
presence of ATP (Hutchison et al., 1993; Stancato et al.,
1993; Dittmar et al., 1997; Pearl and Prodromou, 2006);
thus, inhibitors of ATPase activity, like 17-AAG, a geldana-
mycin derivative, cause dissociation and early ubiquitina-
tion of client proteins (Stebbins et al., 1997; Jez et al.,
2003; Blagg and Kerr, 2006; Powers and Workman,
2006). However, ADP-dependent Hsp90 interaction with
CHORDC1 has been identified and interactions with
some Hsp90-client proteins that constitute core transcrip-
tion machinery have been reinforced by geldanamycin
(Gano and Simon, 2010). In fact, Hsp90, compared with
other chaperones, has less promiscuous interactions
(Picard, 2002; Whitesell and Lindquist, 2005). Extracellu-
lar Hsp90 has been described and found to be correlated
with tumour progression (Eustace and Jay, 2004; Tsut-
sumi and Neckers, 2007; Song et al., 2010). Hsp90 can
also function as a receptor component of known bacterial
virulence factors (Jin et al., 2003; Cabanes et al., 2005;
Reyes-Del Valle et al., 2005; Rechner et al., 2007; Na
et al., 2008). Our finding that cellular Hsp90 interferes
with NadA-mediated adhesion and invasion suggests a
previously unrecognized role for this chaperone during
infection.
Results
Identification of heat shock protein 90 as a
NadA-interacting protein
We performed a NadA affinity column on a membrane-
enriched protein fraction from Chang epithelial cells to
identify eukaryotic cell proteins binding to the adhesin. An
rNadA variant with the membrane-anchor region removed
was covalently bound to a CNBr-activated sepharose
resin and used as stationary phase for affinity chromatog-
raphy. The sample was adsorbed to the column, part of
the eluted bound material was separated by SDS-PAGE
and blotted. The remaining portion was TCA precipitated
and separated in parallel by SDS-PAGE followed by Coo-
massie blue staining.
Hsp90 hampers NadA-mediated infectivity 369
© 2011 Blackwell Publishing Ltd, Cellular Microbiology, 14, 368–385

Far-Western blot, using rNadA as probe, revealed a
specific band with an apparent molecular weight
of ~ 97 kDa in the sample incubated with rNadA
(Fig. 1A, left gel) but not the control (Fig. 1A, right gel).
The corresponding Coomassie-stained bands were
excised from the gel then underwent peptide mass
fingerprint identification using MALDI-TOF TOF mass
spectrometry analysis (Fig. 1B), which revealed exact
matches with Hsp 90, Alpha (a) and Beta (b) isoforms
(Fig. 1C).
Hsp90 binds to NadA in vitro
We confirmed the above results using rNadA and two
sources of recombinant Hsp90 (rHsp90): a commercially
available (Stressgen, ADI-SPP-776) product and an
analogous His-tagged product expressed and purified in
our laboratory. Results of far-Western and dot blot experi-
ments were identical, independent of the source of
rHsp90.
Specific interactions were investigated with SDS-PAGE
separated and blotted rHsp90 and soluble rNadA as
probe (Fig. 2A). Recombinant factor H binding protein
(rfHbp), an unrelated meningococcal surface protein
(Masignani et al., 2003) was used as an internal control.
Binding of bacterial proteins to immobilized rHsp90 was
revealed by anti-NadA and anti-fHbp antibodies. Further
controls included the incubation of membrane blotted
rHsp90 with anti-NadA and anti-fHbp antibodies in the
absence of rNadA and rfHbp (Fig. 2A, lanes 3 and 5
respectively). The position of rHsp90 on the membrane
was assessed by anti-Hsp90 antibodies (Fig. 2A, lane 1).
Immobilized rHsp90 was specifically recognized by rNadA
(Fig. 2A, lane 2) and not by rfHbp (Fig. 2A, lane 4) or the
antibodies against rNadA and rfHbp (Fig. 2A, lanes 3 and
5 respectively).
The potential for binding of rNadA was further tested on
membrane-immobilized rHsp90 in a dot blot experiment.
As shown in Fig. 2B, a nitrocellulose membrane spotted
with rHsp90 and probed with rNadA (Fig. 2B, bottom right)
or rfHbp (Fig. 2B, top right) showed, after extensive wash-
ings, the presence of specifically retained proteins with
anti-NadA antibodies (Fig. 2B, bottom right) or anti-fHbp
antibodies (Fig. 2B, top right). Only rNadA bound to immo-
bilized rHsp90. On the left side of the blots, rNadA and
rfHbp were spotted on the membrane as controls for
anti-NadA (Fig. 2B, bottom left) and anti-fHbp (Fig. 2B,
top left) antibodies.
Specific binding between rNadA and rHsp90 was
tested in a series of co-immunoprecipitation experiments
using rNadA and both rHsp90 and cellular extracts as
sources of Hsp90. The specific recognition of client pro-
teins by Hsp90 is strongly regulated by its ATPase activ-
ity, and ATP/ADP binding and co-chaperone association
drive structural changes that coordinate its association-
release cycle (Pearl and Prodromou, 2006). Inhibition of
Hsp90 ATPase activity causes dissociation and early
ubiquitination of the client proteins (Blagg and Kerr,
2006); 17-AAG, a known Hsp90 inhibitor, specifically
affects the ATP binding pocket (Stebbins et al., 1997;
Jez et al., 2003; Powers and Workman, 2006). Thus, we
analysed the effect of ATP, ADP and 17-AAG, over a
range of concentrations, on the association of NadA
and Hsp90 in parallel with the co-immunoprecipitation
experiments.
Following incubation of the two recombinant proteins
(see Experimental procedures below), recovered immu-
nocomplexes were subjected to Western blot analysis
using anti-Hsp90 antibodies with rfHbp as control
(Fig. 2C). The presence of ADP at a 5 mM concentration
was a crucial step for the formation of NadA–Hsp90
complex in solution (Fig. 2C, top membrane, lane 3). With
sub-optimal concentrations (Fig. 2C, top membrane,
lanes 4 and 5) or without ADP (Fig. 2C, top membrane,
lane 6) no association was detected; no association was
detected between rNadA and rHsp90 in presence of ATP
at any concentration (Fig. 2C, middle membrane). Addi-
tion of 17-AAG 5 mM or more allowed formation of the
NadA–Hsp90 complex (Fig. 2C, bottom membrane), indi-
cating that 17-AAG stabilizes Hsp90 in a conformation
suitable for NadA binding (Grenert et al., 1997; Zhang
et al., 2009). No association of fHbp with Hsp90 was
observed (data not shown).
To verify these findings using Chang cell lysates as a
source of Hsp90, Chang cell total extracts were incu-
bated with rNadA, and anti-NadA antibodies were used
to pull down immunocomplexes. Western blot analysis
revealed that the association of exogenous rNadA with
endogenous Hsp90 was detectable in the presence of
ADP or 17-AAG at the lowest concentrations established
immediately above (Fig. 2D, lanes 3 and 4 respectively)
as well as in the absence of any exogenous compound
(Fig. 2D, lane 5). Of note, NadA co-immunoprecipitated
Hsp90 in the absence of any added nucleotide or
17-AAG (Fig. 2D, lane 5), suggesting that a pool of this
chaperone was potentially in an ADP-like conformation.
The addition of ADP or 17-AAG did not increase the
amount of the complexes detected (Fig. 2D, compare
lane 5 with lanes 3 and 4) but ATP reduced Hsp90
co-immunoprecipitation to the background level (Fig. 2D,
lane 2). rfHbp, shown in absence of exogenously added
compounds, did not associate with Hsp90 (Fig. 2D, lane
6) in this or any other experimental condition studied
(data not shown). Thus, we showed a direct specific
interaction between the cellular chaperone Hsp90 and
the bacterial adhesin NadA and found that an ADP or
17-AAG associated conformation of Hsp90 was required
for this interaction in vitro.
370 P. Montanari et al.

Fig. 1. rNadA binds to a 90 kDa protein from Chang membrane extracts.
A. A protein membrane extract from Chang epithelial cells was loaded on a rNadA-sepharose affinity column and bound species were eluted
with 100 mM NaCl. Three fractions were collected and analysed (1–3). An aliquot of each fraction was loaded in duplicate (left membrane and
right membrane) on SDS-PAGE and transferred to a nitrocellulose membrane. To reveal NadA interacting proteins, the membrane was
overlaid with 20 mg ml-1
of rNadA, revealed with anti-NadA antibodies and HRP-conjugated secondary antibodies (left membrane). The control
membrane was treated with anti-NadA antibodies and HRP-conjugated secondary antibodies (right membrane).
B. The remaining material of the NaCl eluted fractions was TCA precipitated and loaded on SDS-PAGE. The gel was stained with Coomassie
blue and the protein band corresponding to the Western blot positive signal was analysed by peptide mass fingerprint. Only m/z with a
signal/noise ratio above 10 were annotated with the m/z-value and the amino acid position of the corresponding peptide (m/z labelled in black
are common to the sequence of Hsp90a and Hsp90b, m/z labelled in red are specific to Hsp90a, and m/z labelled in blue are specific for
Hsp90b). The asterisks ‘*’ correspond to identified but not annotated signals for Hsp90a (red) and Hsp90b (blue) or both Hsp90a and Hsp90b
(black).
C. Sequence coverage of the Hsp90a and Hsp90b obtained from the peptide mass fingerprint.

rHsp90 speciﬁcally binds to E. coli surface-
exposed NadA
The experiments above established an association of
Hsp90 with recombinant soluble NadAD351–405, which lacks
the anchor region but maintains the trimeric organization
(Magagnoli et al., 2009). We tested the associations
described above using the full-length NadA expressed on
the surface of E. coli BL21(DE3) strain (E. coli–NadA),
which resembles native meningococcal NadA (Capecchi
et al., 2005). We generated a control E. coli–inv from
strain BL21(DE3) carrying the expression plasmid pinv,
coding for the well-known protein invasin from Yersinia
pseudotuberculosis, which conferred an adhesive,
Fig. 2. Characterization of NadA–Hsp90 interaction in vitro.
A. rHsp90 was separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with 20 mg ml-1
rNadA (lane 2) or 20 mg ml-1
rfHbp (lane 4). The presence of the bacterial proteins bound to the membrane was revealed with anti-NadA (lanes 2 and 3) or anti-fHbp
antibodies (lanes 4 and 5) and HRP-conjugated secondary antibodies. Anti-Hsp90 antibodies followed by HRP-conjugated secondary
antibodies were used in lane 1 to detect rHsp90.
B. Two distinct nitrocellulose membranes were spotted with 200 ng of rNadA and 200 ng of rHsp90 (bottom membrane, left and right
respectively) or with 200 ng of rfHbp and 200 ng of rHsp90 (top membrane, left and right respectively) and subsequently incubated with
20 mg ml-1
rNadA (bottom membrane) or 20 mg ml-1
rfHbp (top membrane) respectively. Membrane-bound proteins were revealed with
anti-NadA (bottom membrane) or anti-fHbp (top membrane) antibodies followed by HRP-conjugated secondary antibodies.
C. Equimolar amounts of rNadA and rHsp90 (1 mM each) were incubated for 30 min at 37°C in buffer solutions containing none or increasing
concentrations of either ADP or ATP (50mM–50 mM) or 17-AAG (1–500 mM). The immunocomplexes were pulled down with anti-NadA
antibodies and separated on SDS-PAGE. After transfer to nitrocellulose membranes, detection was performed with anti-Hsp90 antibodies and
HRP-conjugated secondary antibodies. For each panel, in lane 1, 1 mM rHsp90 was loaded as internal control.
D. rNadA (200 ng) (lanes 2–5) was incubated for 30 min at 37°C with Chang cells total extracts alone or in presence of either 5 mM ATP or
5 mM ADP or 5 mM 17-AAG. Revelation of co-immunoprecipitated Hsp90 was performed as described above. In lane 1, rNadA was not added
to the extract. In lane 7, rfHbp was used as unrelated control protein.

invasive phenotype (Isberg and Falkow, 1985; Monack
and Theriot, 2001).
We obtained outer membrane protein (OMP) prepara-
tions from E. coli–NadA (OMP–NadA) and E. coli–inv
(OMP–inv) and checked their binding properties for
rHsp90 by dot blot. The nitrocellulose membranes in
Fig. 3A were spotted with OMP–inv (left membrane, left
side) and rHsp90 (left membrane, right side) or OMP–
NadA (right membrane, left side) and rHsp90 (right mem-
brane, right side), then probed by overlaying OMP–inv
(left membrane) or OMP–NadA (right membrane). Spe-
cific binding to immobilized rHsp90 was detected by anti-
E. coli antibodies. The NadA in OMP–NadA was retained
by the immobilized rHsp90 (Fig. 3A, right membrane, right
side), which did not bind with any protein in the OMP–inv
(left membrane, right side). In another experiment using
OMP obtained from E. coli–pET, a BL21(DE3) carrying
the pET expression vector alone, no binding between
rHsp90 and OMPs was detected (data not shown). This
finding is consistent with a specific interaction between
the E. coli membrane-associated NadA, as present in the
OMP preparation and closer resembling the meningococ-
cal native protein, and rHsp90.
We then performed immunofluorescence analysis to
investigate whether recombinant Hsp90 could bind
directly to the surface of E. coli carrying surface exposed
membrane-anchored NadA. E. coli–NadA and E. coli–inv
were pre-incubated with rHsp90 in PBS buffer for 30 min
at 37°C alone or with: ADP (5 mM), ATP (5 mM) or
17-AAG (5 mM). Following extensive washings, samples
were stained with anti-Hsp90 and revealed with second-
ary antibodies Alexa Fluor 488-conjugated. The bacterial
chromosome was stained with DAPI (4′,6-diamidino-2-
phenylindole). Immunofluorescence microscopy analysis
revealed a significant level of fluorescence, consistent
with binding, associated with E. coli–NadA when incu-
bated with rHsp90 in the presence of ADP and 17-AAG
(Fig. 3B, second and fourth panel from the top), but not in
the presence of ATP or alone (Fig. 3B, first and third panel
from the top). Binding with rHsp90 was specific to E. coli
expressing surface NadA; no rHsp90 associated with
E. coli–inv in any condition tested (see Fig. 3B for incu-
bation with ADP). These results indicate that direct
binding of Hsp90 to NadA occurs with the membrane
anchored protein on the bacterial surface and the speci-
ficity of such an interaction relies on ADP or 17-AAG.
Low levels of Hsp90 strengthen the adhesion and
increase the entry of E. coli–NadA in Chang
epithelial cells
Although it is normally a cytosolic chaperone, Hsp90 has
been reported extracellularly (Tsutsumi and Neckers,
2007), and it can function as a receptor component of
bacterial virulence factors (Jin et al., 2003; Cabanes
et al., 2005; Reyes-Del Valle et al., 2005; Rechner et al.,
2007; Na et al., 2008). To investigate the biological sig-
nificance of the results presented above and to explore
the functional implications of NadA–Hsp90, we investi-
gated the possibility that Hsp90 acts as a NadA receptor
using E. coli expressing NadA and Chang cells in which
Hsp90 was efficiently reduced by RNA silencing. Chang
cells were transfected either with Hsp90-targeted siRNA
or control siRNA (scrambled); Hsp90 reduction at 48 h
was checked by Western blot analysis of total cell lysates
(Fig. 4A). We infected the Hsp90-depleted cells with
E. coli–NadA and E. coli–inv to determine the rate of
adhesion and invasion mediated by NadA and to compare
the effect of decreased Hsp90 levels on a control invasin.
Standard adhesion and invasion values were calculated
by infecting control cells with E. coli–NadA or E. coli–inv.
Adhesion and invasion levels in all tested conditions are
shown in Fig. 4B. Chang cells, transfected with siRNA for
Hsp90 were more susceptible to adhesion (1.8-fold
increase) and invasion (3.8-fold increase) by E. coli–
NadA compared with the control cells transfected with
scrambled siRNA. E. coli–inv adhesive and invasive phe-
notypes were not influenced by Hsp90 silencing. These
results suggest that Hsp90 is unlikely to play a receptor-
like role in the NadA-mediated adhesion and invasion
process, but that total amount of Hsp90 seems to be an
important parameter specifically influencing NadA-
mediated infectivity.
High levels of Hsp90 impairs the infection properties
mediated by NadA
To assess the consequences of E. coli–NadA infection in
Chang epithelial cells under conditions of increased quan-
tity of the chaperone, we transfected Chang cells with a
pQE-TriSystem vector containing hsp90a cDNA (His-
tagged) to overexpress Hsp90 and tested this expression
level by Western blot of total cell lysates (Fig. 5A), com-
pared with control cells transfected with a pQE-TriSystem
empty vector. Hsp90 overexpressing cells and control
cells were infected with E. coli–NadA or E. coli–inv; adhe-
sion or invasion was quantified respectively. The Hsp90
overexpressing cells showed a 30% reduction in E. coli–
NadA adhesion and a 50% reduction in invasion com-
pared with control cells (Fig. 5B). No effects on E. coli–inv
interactions were observed in Hsp90 overexpressing
cells.
Following detection of direct Hsp90 binding to E. coli–
NadA, the effect of exogenously added rHsp90 on NadA-
mediated adhesion and invasion was investigated.
E. coli–NadA and E. coli–inv were pre-incubated with
rHsp90 then added to Chang epithelial cells for adhesion
and invasion assays. The same concentration of

Fig. 3. Binding of rHsp90 to E. coli
membrane-localized NadA.
A. OMP obtained from E. coli expressing
NadA (OMP–NadA) and E. coli expressing
yersinial invasin (OMP–inv) were tested for
their ability to interact with rHsp90 in vitro.
OMP–NadA and rHsp90 (right membrane) or
OMP–inv and rHsp90 (left membrane) were
spotted, separately, on two different
nitrocellulose membranes. The membranes
were individually incubated with 20 mg ml-1
OMP–NadA (right membrane) and 20 mg ml-1
OMP–inv (left membrane); binding was
detected with anti-E. coli specific antibodies,
then HRP-conjugated secondary antibodies.
OMP on the left sides of both membranes
were spotted as an internal control for the
specific antibodies.
B. E. coli–NadA were incubated with
20 mg ml-1
of rHsp90 alone or with the
addition of 5 mM ATP, 5 mM ADP or 5 mM
17-AAG for 30 min at 37°C. After extensive
washings, bacteria were stained with mouse
monoclonal anti-Hsp90 antibodies followed by
Alexa Fluor 488-conjugated antibodies and
DAPI mounting medium to stain bacterial
chromosome. Immunofluorescence allowed to
visualize the rHsp90 bound to bacterial
surface (green) and the bacterial chromosome
(grey).
Binding of rHsp90 to E. coli–inv incubated in
the presence of 5 mM ADP is shown at the
bottom.

exogenous rHsp90 was maintained in the medium
throughout infection. We observed a 50% reduction in the
invasion of E. coli–NadA in the presence of exogenous
rHsp90; NadA-mediated adhesion was slightly increased
under these experimental conditions. E. coli–inv adhesion
and invasion did not appear to be affected by these con-
ditions, suggesting that the effects observed were NadA-
mediated (Fig. 5C).
The inhibition of Hsp90 chaperone activity by 17-AAG
decreases NadA adhesion and invasion
To further examine the relationship between NadA–Hsp90
interaction and NadA-mediated adhesion and invasion,
we used 17-AAG to hamper the nucleotide-induced con-
formational switch that Hsp90 requires for its chaperone
role (Blagg and Kerr, 2006). Chang cells monolayers were
incubated overnight with 17-AAG (0.125, 0.250 and
0.5 mM) before challenge with E. coli–NadA and the
Fig. 4. Hsp90 silencing leads to increased adhesion and invasion
of E. coli–NadA.
A. Chang cells were transfected with control siRNA or Hsp90
siRNA and the level of protein depletion was ascertained by
Western blot of Chang cells total lysates. Beta Actin was used as
loading control.
B. Transfected cells were challenged for adhesion and entry
properties of E. coli–NadA and E. coli–inv (moi 100), results
obtained with Chang cells with silenced Hsp90 are expressed as
fold increase with respect to infection on cells transfected with
control siRNA, arbitrarily considered as 100%. Data represent the
means and standard deviations of several experiments, each
performed in triplicate.
Fig. 5. Interference with adhesion and invasion of E. coli–NadA by
overexpressed and exogenously added Hsp90.
A. Chang cells were transfected with plasmid pQE-TriSystem-
Hsp90a and pQE-TriSystem as control and the level of expression
of Hsp90 was checked by Western blot using Beta Actin as loading
control. Overexpressed Hsp90 holds a His-Tag and localizes at the
highest molecular weight.
B. Transfected cells were infected with E. coli–NadA and E. coli–inv
(moi 100) and the adhesion and entry resulting from cells with
Hsp90 overexpressed were compared with those obtained from cells
transfected with the empty vector (values were arbitrarily ﬁxed at
100%). Values show the means and standard deviations of one rep-
resentative experiment performed in triplicate.
C. E. coli–NadA and E. coli–inv were incubated, separately, with
50 mg ml-1
of rHsp90 for 30 min at 37°C or left untreated. After
incubation, bacteria were used to infect Chang epithelial cells and
rHsp90 (50 mg ml-1
) was maintained during the infection. As a
control, untreated bacteria were used to infect Chang cells in
absence of exogenously added rHsp90. For both, infection was
performed at moi 100. Adhesion and entry of bacteria into cells in
presence of rHsp90 was compared with values obtained with
bacteria infecting cells in absence of exogenously added rHsp90
whose value was established as 100. Means and standard errors of
triplicate samples are shown from one representative experiment.

E. coli–inv control. E. coli–NadA bacterial adhesion was
reduced by 50% to 60% at all concentrations; adhesion of
E. coli–inv was largely unaffected. Invasion of epithelial
cells by E. coli–NadA was inhibited by 50%, 70% and
95%, mirroring the increasing concentration of 17-AAG.
Invasion by E. coli–inv was reduced by 15%, 30% and
55% respectively, using the above mentioned 17-AAG
concentrations (Fig. 6A).
Since prolonged inhibition of Hsp90 by 17-AAG down-
regulates the activity of many Hsp90 client proteins
(Powers and Workman, 2006), protracted exposure
to 17-AAG could have indirectly affected invasion or
adhesion. Therefore, we reduced exposure time while
increasing the concentration of the inhibitor. Chang cells
were pre-incubated with 17-AAG at 0.625, 2.5 and 10 mM
for 1 h prior to bacterial infection, that was performed
maintaining the indicated concentrations of the inhibitor.
Representative results are shown in Fig. 6B. Adhesion
and invasion by E. coli–NadA were reduced approxi-
mately 20% and 60%, respectively, whereas E. coli–inv
adhesion and invasion decreased by 10% or less. To
verify if intracellular pathways assisted by Hsp90 were
affected by short-term treatment with 17-AAG, we
checked the level of expression of Akt, a representative
Hsp90 client protein (Powers and Workman, 2006), by
Western blot. With short-term incubation at high 17-AAG
concentrations, Akt amount was unaffected by this
Hsp90 inhibitor suggesting that cellular processes
Fig. 6. 17-AAG treatment negatively
interferes with the adhesion and invasion
exerted by NadA.
A. Chang cells were treated with different
concentrations of 17-AAG during an overnight
incubation, then infected with E. coli–NadA
and E. coli–inv at an moi of 100.
B. Chang cells were incubated with different
doses of 17-AAG for 1 h and then submitted
to E. coli–NadA and E. coli–inv infection (moi
100).
For all experiments, adhesion and entry of
bacteria into treated cells were compared with
those into untreated cells, whose value was
established as 100. Data represent the
means Ϯ standard deviations of several
experiments performed in triplicate. 17-AAG,
at the indicated concentrations, was
maintained during the infection.

indirectly assisted by this chaperone were not hampered
(data not shown).
While our results suggest that the first line of Hsp90
interference with NadA-mediated adhesion and invasion
is independent of any action on client proteins, this con-
clusion would not rule out a requirement for indirect
mechanisms, which could be shared by other pathogens
and could confirm the reduced invasion of E. coli–inv after
prolonged periods of inhibitor treatment.
Hsp90 inhibitors strongly induce Hsp90 expression by
activating Hsf1 (heat shock factor 1), an essential hsp90
transcription factor (Whitesell et al., 2003). We used
Western blot analysis to estimate the amount of Hsp90 in
Chang cells lysates following 17-AAG treatment over-
night at concentrations of 0.125, 0.25 and 0.5 mM or for
5 h at concentrations of 0.625, 2.5 and 10 mM; these
were the same exposure times used to generate the data
in Fig. 6. A representative result is in Fig. 7. In Chang
cells, Hsp90 levels increased after 17-AAG treatment
(Fig. 7, compare with lane 1), and Hsp90 induction over
the basal level (Fig. 7, lane 1) was observed following
17-AAG exposure, both overnight (Fig. 7, lanes 2–4) and
for 5 h (Fig. 7, lanes 5–7). Western blot indicated that the
concentration of 17-AAG did not affect Hsp90 levels in
these time frames, although longer incubation periods
led to stronger expression.
Hsp90 colocalizes with unencapsulated meningococcal
strains during infection of Chang cells
Escherichia coli expressing NadA allows the analysis
of the specific contribution of this adhesin/invasin in
meningococcal host interaction because the diverse
N. meningitidis virulence factors have likely redundant
functions (Virji, 2009). To assess possible interactions
between cellular Hsp90 and meningococci after infec-
tion, we conducted confocal analysis of Chang cells
infected with two unencapsulated meningococcal
strains: a nadA knockout mutant, MC58 SiaD-
/NadA-
that carries a truncated form of nadA gene and lacks the
expression of the protein (Capecchi et al., 2005) and
strain MC58 SiaD-
/NadA-
/cNadA that overexpresses
NadA by mean of genetic complementation as described
in Experimental procedures. In the MC58 SiaD-
/NadA-
/
cNadA strain, nadA is controlled by a constitutive pro-
moter (Ptac) and NadA is produced at a higher extent
compared with the wild-type strain, which allowed us to
overcome the low basal expression of NadA and poten-
tial phase-variability.
Chang cells were infected for 6 h with the two menin-
gococcal strains then underwent fixation, permeabiliza-
tion and staining. A representative confocal analysis is
shown in Fig. 8. Bacteria penetrated into cells (Fig. 8,
bottom panels), generally in close proximity to Hsp90;
the overexpressing strain is shown in Fig. 8A and the
knockout strain in Fig. 8B. Hsp90 appeared to form intra-
cellular cluster-like structures that extended around the
meningococci; however, the apical surface of infected
cells revealed marked differences between the strains.
Cells infected with the nadA knockout strain showed
many clustered bacteria with very limited colocalization
with Hsp90 (Fig. 8B, top panels), while the NadA over-
expressing strain was rarely found on the cell surface.
Clusters of the NadA overexpressing strain were com-
pletely surrounded by Hsp90 (Fig. 8A, top panels). Thus,
NadA specifically affected interaction of Hsp90 with
adhered meningococci. The relevance of this finding
deserves further investigation.
Discussion
In the course of investigations of cellular interactions with
NadA, a meningococcal adhesin and invasin, we identi-
fied a new, non-canonical role for Hsp90, as a line of
interference with NadA-mediated activity. This role
appeared independent of any action on Hsp90 client pro-
teins. We provided robust evidence of in vitro associations
between NadA and Hsp90 by ligand overlay assay, dot
blots, co-immunoprecipitation and immunofluorescence.
The similarity of the specific Hsp90 binding properties for
both soluble and anchored NadA was also demonstrated.
Further, the association between Hsp90 and NadA was
found to be finely tuned by chaperone-binding small mol-
ecules in vitro. While NadA associated with the ADP-
bound conformer of Hsp90 and 17-AAG-bound Hsp90, in
the absence of nucleotides or in the presence of ATP, no
Fig. 7. 17-AAG induces Hsp90 expression.
Chang cells lysates were tested for the level
of expression of Hsp90 by Western blot after
both an overnight (O/N) and a 5 h treatment
with different concentrations of 17-AAG. Beta
Actin was used as loading control.

Fig. 8. Confocal laser scanning microscopy to assess interactions of N. meningitidis with Hsp90.
A and B. Confluent Chang cells monolayers grown on coverslips were infected with the MC58 SiaD-
/NadA-
/cNadA (A) or MC58 SiaD-
/NadA-
(B) strains at an moi of 100. After 6 h, non-adherent bacteria were washed off, subsequently cells were fixed and then permeabilized.
Meningococci (Men B) and Hsp90 were stained as described in Experimental procedures, DAPI was used to stain nuclei and bacterial
chromosome, phalloidin stained F-actin (Men B, red; Hsp90, green; DAPI, blue; F-actin, grey). As indicated, for each x-z section an x-y
projection is extracted from both the apical and the basal surface of infected cells. Magnification of x-y fields are shown on the right. Phalloidin
was omitted from magnifications for clarity.

association between NadA and Hsp90 was detected.
Either ADP or 17-AAG was also necessary for specific
binding of Hsp90 to E. coli expressing surface NadA.
Previous identification of surface Hsp90 and Gp96, the
endoplasmic reticulum homologue of Hsp90, as patho-
gen protein binding factors was made by ligand overlay
(Jin et al., 2003; Cabanes et al., 2005) or immunoblotting/
pull down assay (Reyes-Del Valle et al., 2005; Rechner
et al., 2007; Na et al., 2008). We used an analogous
approach to identify the association of NadA to Hsp90.
Interactions of Hsp90 with client proteins are less promis-
cuous than those of other chaperones because of its
involvement with proteins that maintain the structural
integrity of essential regulators of cellular homeostasis
(Picard, 2002; Whitesell and Lindquist, 2005). Associa-
tion of Hsp90 to client proteins requires ATP as co-factor
whereas hydrolysis to ADP induces dissociation of the
complexes (Hutchison et al., 1993). Surprisingly, using
recombinant proteins in vitro, we discovered that Hsp90
binding to NadA required ADP or 17-AAG and was inhib-
ited by ATP. These results were inconsistent with the
previous understanding of Hsp90. Further, NadA was
able to co-immunoprecipitate Hsp90 from a crude cell
extract, with the unanticipated result that Hsp90 binding
with rNadA was then independent of any added nucle-
otide. Co-factors and co-chaperones in the cell lysate
could have played a role in this result, counteracting the
high concentration of cellular ATP relative to ADP, which
was shown to be necessary in other experiments. Alter-
natively, an adequate amount of cellular Hsp90 could
have been bound to ADP to produce this result. In all
tested conditions, however, the addition of ATP caused
Hsp90–NadA complex dissociation while exogenous ADP
or 17-AAG allowed complex formation. NadA binding to
the ADP or 17-AAG form of Hsp90 suggests a function for
this chaperone outside of its classical ATP-dependent
binding and ATP-hydrolysis release of client proteins.
Reassuringly, our finding is supported indirectly by recent
work that identifies ADP-dependent Hsp90 interaction
with CHORDC1 and several additional client protein inter-
actions that are reinforced by geldanamycin (Gano and
Simon, 2010).
To isolate the role of NadA as a single meningococcal
factor, we exploited a heterologous well-defined bacterial
carrier, E. coli, which is able to correctly fold and carry
surface NadA (Capecchi et al., 2005). This system
allowed us to perform adhesion/invasion assays whose
outcome could be directly ascribed to NadA. In infection
assays with Chang epithelial cells, we found evidence of
a role of Hsp90 interfering against NadA-driven infection.
Lower cellular levels of Hsp90 allowed greater NadA
attachment and massive invasion of E. coli bearing
surface-exposed NadA. Conversely, plasmid-driven over-
expression of Hsp90 reduced E. coli–NadA adhesion and
entry. The addition of exogenous rHsp90 into the infection
system led to a substantial decrease in the invasion medi-
ated by NadA supporting the hypothesis of an interference
effect by the chaperone.
To evaluate the interaction between Hsp90 and NadA
in the meningococcus we carried out a confocal micros-
copy analysis of Chang epithelial cells infected with
either a nadA knockout strain or a NadA overexpressing
strain. Results indicated that Hsp90 colocalized with
intracellular bacteria for both strains. However, differ-
ences between extracellular and intracellular bacteria
were pronounced. Specifically, many fewer NadA over-
expressing meningococci were identified extracellularly
when compared with nadA null mutants; moreover, the
former were completely surrounded by Hsp90 whereas
the latter were merely colocalized. This observation
could be explained by two paradoxical mechanisms:
accelerated intracellular localization of the NadA overex-
pressing strain or a faster kinetics of association/
dissociation from the cell surface for Hsp90 coated
bacteria. Although the full significance of this observation
remains to be demonstrated, our analysis suggests that
Hsp90 binding could represent a redundant function
shared by other meningococcal factors. Nevertheless,
the presence of NadA could fine-tune the process of
infection so that Hsp90 becomes crucial. Studies are
ongoing to clarify our findings.
One important consideration for our work is that the
invasion and adhesion mechanisms of NadA remain
largely unelucidated, a circumstance which in fact
prompted the initial experiments reported here. An inter-
esting finding was that the inhibition of NadA, a surface
protein belonging to an obligate human pathogen, by
Hsp90 appeared to be dependent on the amount of each
protein present, which raises the question whether men-
ingococcal strains without NadA will bind to Hsp90. Our
results suggest diverse associations with a nadA knock-
out strain; however, further experimentation using strains
that lack the nadA gene would help clarify this relation-
ship. Interestingly, NadA is present on relatively few men-
ingococcal strains as compared with other surface
proteins like PorA or fHbp. Evolutionary pressure could
gradually have eliminated NadA-harbouring strains
because human cell factors evolved to recognize NadA
and mount immune responses against it, yet an associa-
tion of NadA with pathogenic isolates has been noted
(Comanducci et al., 2002; 2004). Another area requiring
further investigation is the phase variation of NadA, which
has been indirectly suggested in other contexts (Metruc-
cio et al., 2009). As a matter of fact, we ignore if NadA is
selectively expressed during critical steps of meningococ-
cal pathogenesis.
The Hsp90 inhibitor 17-AAG is another co-factor that
allowed binding of Hsp90 to NadA. Members of this

drug-derivative family mimic nucleotides and hamper
ADP/ATP exchange as well as intrinsic Hsp90 ATPase
activity preventing the chaperone from assisting its
client proteins (Powers and Workman, 2006; Workman
et al., 2007). We showed that short-term exposures
to 17-AAG specifically interfered with NadA-mediated
infection, while long-term Hsp90 inhibition affected
invasion processes mediated by yersinial inv, our control
invasin. We speculate that chemical inhibition by
Hsp90 hinders NadA invasion in two additive ways: a
background, 17-AAG-dose-dependent inhibition towards
putative common mechanisms relevant for bacterial
invasion, such as endocytosis or cytoskeleton remodel-
ling (Yang et al., 2004; Amiri et al., 2007), and a specific
NadA–Hsp90 interaction-related process relying on
the availability of Hsp90 species appropriate to bind
NadA. Increased protein expression of Hsp90 after
17-AAG treatment appeared homogeneous and dose-
independent within a single incubation time frame. We
hypothesize that a physical association between menin-
gococcal NadA and Hsp90 interferes with bacterial
attachment to, and invasion of, Chang cells, in a manner
dependent on the relative amounts of these proteins.
Thus, the NadA adhesive phenotype appears unaffected
by background side-effects. Further, nucleotides or
co-chaperones might participate in association/
dissociation cycles between NadA and Hsp90 regulating
such phenomena, should they be found to occur. Since
extracellular Hsp90 has been identified (Eustace and
Jay, 2004; Tsutsumi and Neckers, 2007; Song et al.,
2010), this would support different roles for this chaper-
one in intra- and extracellular contexts and could there-
fore be of use in further interpretation of the results we
presented above. Yet, while the confocal microscopy
analysis of Chang epithelial cells suggests that extracel-
lular Hsp90 may bind NadA overexpressing meningo-
cocci and showed that intracellular Hsp90 colocalized
with both nadA knockout and overexpressing meningo-
coccal strains, confirmatory data are required. Our
results also suggest that Hsp90 binding could represent
a redundant function shared by other meningococcal
factors and that examining the interactions of Hsp90
with NadA could help identify areas in the infectious
process where Hsp90 represents a crucial component.
Cellular Hsp90 might play a role in the context of innate
immune mechanisms by preventing the meningococcal
NadA from promoting cell adhesion and invasion.
Our results demonstrated NadA–Hsp90 interaction that
relied on specific cofactors such as ADP or 17-AAG, and
the displacement of such interaction was driven by ATP.
Based on these observations, it would be of interest to
further characterize the NadA infection process under
conditions of impaired ADP/ATP ratio, as during altered
energy balance.
Experimental procedures
All procedures were performed following appropriate ethical
guidelines for the treatment of laboratory animals whenever
applicable and good laboratory practice.
Cell cultures
Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4,
human conjunctiva, ATCC CCL-20.2) were maintained in Dulbec-
co’s modified Eagle’s medium (DMEM) supplemented with
15 mM L-glutamine, antibiotics and 10% heat-inactivated FBS
(FBSi). Cells were grown at 37°C in 5% CO2.
Bacterial strains, growth conditions and
OMP preparation
Escherichia coli BL21(DE3) (Novagen) was used to express
genes coding for full-length NadA and NadAD351–405 as previously
described (Capecchi et al., 2005).
The expression of Y. pseudotuberculosis invasin in E. coli was
obtained by transforming the plasmid pinv (Monack and Theriot,
2001), a generous gift from Professor Monack (Stanford Univer-
sity School of Medicine, Stanford, CA, USA), into E. coli
BL21(DE3).
Escherichia coli was cultured at 37°C in Luria–Bertani (LB)
broth supplemented with 100 mg ml-1
ampicillin (E. coli–NadA) or
30 mg ml-1
chloramphenicol (E. coli–inv). Protein expression for
full-length NadA and yersinial invasin was achieved without addi-
tion of IPTG (uninduced conditions), exploiting expression due to
leakage of the induction system.
Meningococcal MC58 SiaD-
/NadA-
and MC58 SiaD-
/NadA-
/
cNadA were serogroup B strains. Unencapsulated MC58
SiaD-
/NadA-
as previously described (Capecchi et al., 2005)
lacks NadA expression. To achieve complementation of
NadA a copy of the nadA gene was inserted under the
control of the Ptac promoter in the non-coding region of the
MC58 SiaD-
/NadA-
chromosome between the converging
open reading frames NMB1428 and NMB1429. The plasmid
for complementation of the nadA null mutant, pCOMnadA, was
previously described (Tavano et al., 2009) and was used to
transform the MC58 SiaD-
/NadA-
strain to generate the
MC58 SiaD-
/NadA-
/cNadA strain. The restoration of NadA
expression on the surface of the complemented strain was
confirmed by Western blot and FACS analysis. NadA was pro-
duced in a trimeric form and expressed on the surface of MC58
SiaD-
/NadA-
/cNadA at a higher extent compared with MC58
SiaD-
, the isogenic unencapsulate wild-type strain (data not
shown).
Outer membrane proteins (OMPs) were recovered from
E. coli strains on the basis of Sarkosyl insolubility following
the rapid procedure described by Carlone et al. (1986). Briefly,
bacteria were harvested, suspended in 1 ml of 10 mM Hepes
buffer (pH 7.4) and sonicated on ice. Cell membranes were
recovered by successive centrifugations at 15 600 g at 4°C
in a microcentrifuge. Cytoplasmic membranes were solubilized
by addition of an equal volume of 2% Sarkosyl in 10 mM
Hepes (pH 7.4). The outer membranes were then recovered by
centrifugation and resuspending the pellet in 10 mM Hepes
buffer.

Purified proteins, antibodies and reagents
Recombinant NadAD351–405 was purified according to previously
described procedures (Capecchi et al., 2005). Recombinant fHbp
(variant 1) was obtained as previously described (Masignani
et al., 2003). Recombinant Hsp90 was purchased from Stress-
gen (ADI-SPP-776). Recombinant His-tagged Hsp90 was
expressed by an E. coli BL21(DE3) strain transformed with the
plasmid pDEST14Hsp90, provided by the Protein Science Group
at Novartis Institute for Biomedical Research (Emeryville, CA,
USA). Recombinant His-tagged Hsp90 was purified as follows:
one single colony of E. coli BL21(DE3) strain expressing Hsp90-
His was inoculated in LB + ampicillin and grown overnight at
37°C, diluted in fresh LB medium and grown at 37°C to an OD of
0.6–0.8. The protein overexpression was induced by the addition
of 1 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG; Sigma)
for 3 h. Recombinant Hsp90 6¥ His fusion protein was purified by
affinity chromatography on Ni2+
-conjugated chelating fast-flow
Sepharose (Pharmacia). The purity was checked by SDS-PAGE
electrophoresis staining with Coomassie blue. The protein
content was quantified by Bradford reagent (Bio-Rad).
The mAb (9F11) recognizing NadA was produced by immuniz-
ing 4- to 6-week-old female CD1 mice with 20 mg of NadA recom-
binant protein (allele 3) administered intraperitoneally together
with complete Freund’s adjuvant (except for the third dose,
which was administered without adjuvant). Three days later,
the mice were sacrificed and their spleen cells were fused
with myeloma cells (P3 ¥ 63-Ag8.653) at a ratio of five spleen
cells to one myeloma cell. After a 2-week incubation
in hypoxanthine-aminopterin-thymidine selective medium, the
hybridoma supernatants were screened for antibody binding
activity by enzyme-linked immunosorbent assays (ELISAs).
The mouse polyclonal anti-serum against fHbp had been
obtained previously (Masignani et al., 2003). The mAb (AC88)
against Hsp90 was purchased from Stressgen (SPA-830).
The rabbit polyclonal anti-serum against Hsp90 was obtained
by immunizing a New Zealand White rabbit with 25 mg of recom-
binant His-tagged Hsp90. The recombinant protein was given
subcutaneously with Freund’s incomplete adjuvant for the first
dose and with Freund’s complete adjuvant for the second (day
21) and the third (day 35) doses. A blood sample was taken on
day 49. Finally, the serum was purified by affinity chromatography
on CNBr activated Sepharose 4B resin (Pharmacia) according to
the manufacturer’s instructions. Rabbit polyclonal anti-E. coli
serum (DAKO) was used to recognize E. coli OMPs. The mAbs
(AC74) against Beta Actin was purchased from Sigma (A2228,
Sigma). Rabbit polyclonal anti-Men B OMVs antibodies were
obtained as previously described (Giuliani et al., 2006). Poly-
clonal Goat Anti-Mouse or Anti-Rabbit Immunoglobulins/HRP
were purchased from DAKO. Alexa Fluor 488 goat anti-mouse
IgG, Alexa Fluor 568 goat anti-mouse IgG, Alexa Fluor 647 goat
anti-rabbit IgG and Alexa Fluor 488-conjugated phalloidin were
from Molecular Probes.
17-AAG (17-N-allylamino-17-demethoxygeldanamycin), ADP
and ATP were resuspended, stored and implied in accordance
with the manufacturer’s specifications (Sigma).
Identification of Hsp90 by affinity chromatography
About 2 ¥ 108
monolayered Chang cells were detached using
CDS solution (Sigma), washed with PBS and lysed using hypo-
tonic solution (10 mM NaCl, 10 mM Tris-base, 0.2 mM CaCl2,
1.5 mM MgCl2) supplemented with complete protease inhibitor
(Roche) for 40 min at 4°C in rotation. A pellet was collected by
centrifugation at low speed 3000 g for 5 min. Membrane proteins
were extracted from the pellet in 2% Brij 96 in 50 mM Hepes
pH 7.4, 150 mM NaCl and the complete protease inhibitor and
sample were submitted for two subsequent centrifugations at
21 000 g for 10 min and at 190 000 g for 20 min. Supernatant
was recovered, diluted 1:2.5 with 50 mM Hepes pH 7.4 contain-
ing complete protease inhibitor to equilibrate in 0.8% Brij 96 and
60 mM NaCl. The sample was pre-cleared on a deactivated
CNBr-activated Sepharose 4-Fast Flow resin, pre-equilibrated in
the same buffer, for 1 h at 4°C. The pre-cleared material was then
loaded on CNBr-activated Sepharose 4-Fast Flow resin coupled
with rNadA pre-equilibrated in 0.8% Brij 96 in 50 mM Hepes
pH 7.4, 60 mM NaCl and complete protease inhibitor. The bound
material was eluted with 100 mM NaCl in 50 mM Hepes, and
0.8% Brij 96 collecting three fractions. For each fraction, about
1/50 of the total volume was checked in ligand overlay assay
(far-Western blot) for the presence of specific bands recognized
by rNadA protein. The remaining material of each fraction was
TCA precipitated and loaded on a parallel identical gel stained
with Coomassie blue.
In-gel protein digestion and MALDI-TOF TOF mass
spectrometry analysis
Protein spots were excised from the gels, washed with 50 mM
ammonium bicarbonate, acetonitrile (50:50, v/v), washed once
with pure acetonitrile, and air-dried. Dried spots were digested for
2 h at 37°C in 12 ml of 0.012 mg ml-1
sequencing grade modified
trypsin (Promega) in 5 mM ammonium bicarbonate. After diges-
tion, 0.6 ml was loaded on a matrix PAC target (Prespotted
AnchorChip 96, set for proteomics, Bruker Daltonics) and air-
dried. Spots were washed with 0.6 ml of a solution of 70%
ethanol, 0.1% trifluoroacetic acid. Mass spectra were acquired on
an Ultraflex MALDI-TOF TOF mass spectrometer (Bruker Dalton-
ics) in reflectron, positive mode in the mass range of 900–
3500 Da. Spectra were externally calibrated by using a
combination of standards prespotted on the target (Bruker Dal-
tonics). MS spectra were analysed with flexAnalysis (flexAnalysis
version 2.4, Bruker Daltonics). Monoisotopic peaks were anno-
tated with flexAnalysis default parameters and manually revised.
Protein identification was carried from the generated peak list
using the Mascot program (Mascot server version 2.2.01, Matrix
Science). Mascot was run on a public database, National Center
for Biotechnology Information non-redundant (NCBInr).
Detergent lysis of Chang cells
For immunoprecipitation using Chang cells total extracts and for
analysis of protein level expression, cells were lysed on ice in
RIPA buffer (Sigma) supplemented with complete protease inhibi-
tor (Roche) for 30 min. Cell debris was removed by centrifugation
at 14 000 g for 15 min. In some cases, sonication was performed
to increase yields.
SDS-PAGE, Western blotting and ligand overlay assay
All SDS-PAGE reagents were purchased from Invitrogen. Equal
amounts of proteins were prepared in 4¥ NuPAGE LDS Sample

Preparation Buffer and 10¥ NuPAGE Sample Reducing Agent
and separated on NuPAGE polyacrylamide gels using NuPAGE
SDS Running Buffers. Proteins were transferred to nitrocellulose
membranes for Western blot analysis. Membranes transferred
(for Western blot) or spotted (for dot blot) proteins were blocked
with PBS containing 0.05% Tween 20 (PBST) + 5% dried skim
milk at room temperature for 1 h. For dot blot and far-Western
blot analysis, prior to primary antibodies incubation, membranes
were overlaid with PBS containing 25 mg ml-1
of recombinant
overlaying proteins or 25 mg ml-1
of OMPs at 4°C for 4 h.
After extensive washings in PBST, proteins bound on nitro-
cellulose membranes were detected with specific primary
antibodies followed by the corresponding HRP-conjugated sec-
ondary antibodies.
Co-immunoprecipitations
Dynabeads®
protein G (Invitrogen) were incubated with anti-
NadA monoclonal mouse antibodies (9F11) in PBS + 0.1%
Tween 20 for 40 min at room temperature. Extensive washings
with PBS + 0.1% Tween 20 were performed to eliminate
excess antibodies. Equimolar amounts (1 mM) of rNadA and
rHsp90 were incubated for 30 min at 37°C in immunoprecipita-
tion buffer (10 mM MgCl2, 300 mM KCl, 2.5% Triton X-100 in
PBS) with 1 mM DTT. Nucleotides or 17-AAG were added to
the incubation mixture at the concentration specified in each
legend, ranging from 50 mM to 50 mM for ADP and ATP and
1 mM to 500 mM for 17-AAG. Samples were incubated with
anti-NadA antibodies pre-loaded magnetic beads in immuno-
precipitation buffer in presence of 1 mM DTT and gently rotated
for 30 min at room temperature. After removal of the superna-
tant, beads were washed three times with PBS + 1 mM DTT.
Protein complexes were recovered by adding SDS-PAGE
sample buffer including reducing agent and boiling for 5 min at
100°C. Samples underwent SDS-PAGE and Western blot
analysis. The presence of co-immunoprecipitated Hsp90 on
the membrane was revealed with rabbit polyclonal anti-
Hsp90 antibodies followed by specific HRP-conjugated
antibodies.
Total extracts from Chang cells, used as source of Hsp90
for co-immunoprecipitation experiments, were prepared as
described above. Cellular extracts from 106
cells were incubated
with 200 ng of rNadA for 30 min at 37°C under the conditions
described above. Co-immunoprecipitated Hsp90 was revealed
as detailed above.
Immunofluorescence analysis
For demonstration of specific coating of rHsp90 onto E. coli
expressing NadA, E. coli–NadA and E. coli–inv strains were sus-
pended in PBS and rHsp90 added to a final concentration of
20 mg ml-1
in the presence or absence of either 5 mM ATP or
5 mM ADP or 5 mM 17-AAG. After incubation with gentle mixing
for 30 min at 37°C, bacteria were washed extensively with PBS
and spread on polylysine-coated plates. Samples were then fixed
in 3.7% paraformaldehyde, washed and blocked with PBS + 3%
Bovine Serum Albumin (BSA) (Sigma) +10% Normal Goat Serum
(Invitrogen) for 1 h at room temperature. After multiple washings,
samples were incubated with mouse monoclonal anti-Hsp90 anti-
bodies (1:100) for 1 h at room temperature. Washings to remove
unbound primary antibodies were followed by incubation with
Alexa Fluor 488 goat anti-mouse IgG (1:400). Labelled prepara-
tions were mounted with ProLong®
Gold antifade reagent with
DAPI (Molecular Probes) and analysed with a Zeiss LSM-710
confocal microscope.
Measurement of bacterial association and invasion by
viable counting
Chang cells were seeded on 24-well tissue culture plates (1 ¥ 105
cells per well), and after 24 h of incubation in an antibiotic-free
medium, approximately 3 ¥ 107
[multiplicity of infection (moi) of
100:1] bacteria were added per well in DMEM supplemented with
1% FBSi and incubated for 4 h at 37°C in 5% CO2. After removal
of non-adherent bacteria by washing, cells were lysed with 1%
saponin (Sigma), DMEM + 1% FBSi was used to harvest bacte-
ria, and serial dilutions of the suspension were plated onto LB
agar to calculate the number of colony-forming units. To deter-
mine the number of intracellular bacteria, infected Chang mono-
layers were treated with gentamicin (200 mg ml-1
) for 1 h at 37°C.
After washing, cells were lysed and the bacteria recovered and
plated.
To test the effect of exogenously added rHsp90, E. coli–NadA
and E. coli–inv were suspended in DMEM + 1% FBSi containing
50 mg ml-1
of rHsp90 and incubated with gentle mixing for 30 min
at 37°C. Bacteria were then used to infect Chang epithelial cells;
rHsp90 was maintained in the medium throughout the infection
period.
To test the effect of Hsp90 chaperone activity inhibition, cells
were pre-incubated either for an overnight period (approximately
15 h) or for 1 h at 37°C before infection with different concentra-
tions of 17-AAG, which remained constant throughout the infec-
tion period. No effect on bacterial or cell viability at the
concentrations and times used was observed (data not shown).
Cell transfections: Hsp90 siRNA and overexpression
To silence gene expression by siRNA, Chang cells were trans-
fected either with a mixture containing four independent siRNA
constructs (2.5 nM each, sc-35608, Santa Cruz Biotechnology)
directed to both hsp90a and hsp90b or with a scrambled con-
struct siRNA (10 nM, sc-44230, Santa Cruz Biotechnology), as
negative control, using HiPerfect Transfection Reagent (Qiagen)
according to the manufacturer’s instructions. Chang cells were
infected with E. coli–NadA or E. coli–inv 48 h after transfection.
For overexpression experiments cells were transfected either
with 0.5 mg of pQE-TriSystem vector containing hsp90a cDNA
and encoding for an additional 10X His-Tag, or with 0.5 mg of
pQE-TriSystem empty vector, as a negative control (both from
QIAgenes Expression Kit, Qiagen) using FuGene 6 reagent
(Roche) as recommended by the manufacturer. Infection assays
were performed 24 h after transfection. All plasmid DNA was
prepared using the EndoFree Plasmid Maxi Kit (endotoxin free;
Qiagen).
Confocal microscopy
Dual labelling of Hsp90 and meningococci was performed in a
series of steps. Chang epithelial cells were seeded on coverslips

(1 ¥ 105
cells per coverslip) and grown to confluency. The day
of infection the culture medium was removed and fresh
DMEM + 1% FBSi added. Bacteria grown on GC agar plates
were washed in PBS once, resuspended in DMEM + 1% FBSi
and then added (~3 ¥ 107
bacteria per coverslip, moi of 100:1) to
monolayers. Cells and bacteria were incubated for 6 h at 37°C in
5% CO2. After removal of non-adherent bacteria by washing,
samples were fixed in 2% paraformaldehyde, washed and then
permeabilized using PBS + 0.l% Triton X-100 + 1% saponin for
20 min at room temperature. Monolayers were washed and
blocked with PBS + 0.1% Triton X-100 + 3% BSA + 10% Normal
Goat Serum for 1 h at room temperature, then washed and
incubated with: (i) rabbit polyclonal anti-Men B OMVs antibodies
(1:500) followed by Alexa Fluor 647 goat anti-rabbit IgG (1:400)
to detect meningococci and (ii) mouse monoclonal anti-Hsp90
antibodies (1:1000) followed by Alexa Fluor 568 goat anti-mouse
IgG (1:400) to detect cellular Hsp90. Each antibody was diluted in
PBS + 0.1% Triton X-100 + 1% BSA, and incubations took place
at room temperature. Alexa Fluor 488-conjugated phalloidin
(1:200) was used together with secondary antibodies to stain
F-actin. Glass coverslips were mounted with ProLong®
Gold anti-
fade reagent with DAPI and analysed with a Zeiss LSM-710
confocal microscope.
Acknowledgements
We thank Marialina Bernardini (Università La Sapienza, Roma)
and Marco Soriani (Novartis Vaccines and Diagnostics) for very
helpful discussions. Professor Monack of Stanford University
generously provided a plasmid, as described above. Lisa DeTora
(Novartis Vaccines and Diagnostics) is gratefully acknowledged
for providing editorial guidance and support. We are grateful to
Giorgio Corsi for artwork and Mirko Cortese for technical help.
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