INFECTION AND IMMUNITY, June 2009, p. 2285–2293
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 6
Role of HrpA in Biofilm Formation of Neisseria meningitidis and
Regulation of the hrpBAS Transcripts?†
R. Brock Neil and Michael A. Apicella*
Department of Microbiology, 3-401 BSB, University of Iowa, Iowa City, Iowa 52242
Received 9 December 2008/Returned for modification 29 January 2009/Accepted 4 March 2009
Two-partner secretion systems of gram-negative organisms are utilized in adherence, invasion, and biofilm
formation. The HrpAB proteins of Neisseria meningitidis are members of a two-partner secretion system, and
HrpA is established as being important to adherence and intracellular escape. This study set out to determine
the expression pattern of members of the hrpBAS putative operon and to find a functional role for the HrpA
protein. The upregulation of these genes was found in situations of anaerobiosis and cell contact. These
observations prompted the study of the function of HrpA in biofilms on human bronchial epithelial cells. HrpA
mutants in encapsulated and unencapsulated NMB strains demonstrated biofilm growth equivalent to that of
the wild-type strain at 6 h but a decreased ability to form biofilms at 48 h. Biofilms formed by hrpA mutants
for 48 h on collagen-coated coverslips demonstrated significant reductions compared to those of wild-type
strains. Taken together, these observations imply a role for HrpA in the biofilm structure. Further analysis
demonstrated the presence of HrpA on the surface of the bacterium.
Neisseria meningitidis is the causative agent of meningococ-
cal meningitis. It is a common isolate of the respiratory tract,
living as a commensal in 5 to 10% of the population during
periods in which infection is endemic (4). The primary adhesin
for N. meningitidis is the type IV pilus, which is utilized as a
long-range adherence factor. The pilus retracts after attach-
ment to the host tissue, resulting in the intimate contact of the
bacterium with the host respiratory tissue (53). Nonfimbrial
adhesins such as Opa, Opc, NhhA, and NadA are important
for intimate adherence to the host membrane (3, 41, 52).
Recently, a type V secretion system was described in N.
meningitidis (42, 46, 48). There are two categories of type V
secretion in gram-negative organisms. The first is the auto-
transporter, and the second is the two-partner secretion sys-
tem. Both utilize the Sec transport system to cross the inner
membrane. The autotransporter has its own secretion domain
that embeds in the outer membrane and catalyzes its own
secretion across the outer membrane (20). The two-partner
system consists of two proteins generically referred to as TpsA
and TpsB, in which the transporter TpsB embeds in the outer
membrane and serves as a dedicated transporter for TpsA
(27). This secreted protein often is a large adhesin. The best-
characterized two-partner secretion system is the FhaBC sys-
tem in Bordetella pertussis, in which the adhesin, FhaB, is se-
creted by the outer membrane transporter, FhaC (10, 17).
FhaB is processed at the N-terminal end when it passes
through the Sec pathway in the inner membrane and is pro-
cessed again at the C-terminal end as it exits the outer mem-
brane by SphB1, an autotransporter subtilisin-like protease (8,
22). There is some evidence that proteases in addition to
SphB1 are involved in the final processing event (30). Com-
pletely processed FhaB is referred to as FHA. Common char-
acteristics of the TpsA adhesins are a high molecular weight,
an extended signal peptide, a predicted beta-solenoid shape for
at least one domain of the protein, and a hemagglutination
domain (2, 6, 22, 27).
The N. meningitidis two-partner secretion system consists of
HrpAB, where HrpA is the putative adhesin and HrpB is the
outer membrane transporter. HrpB secretes HrpA, which
functions as an adhesin in an unencapsulated and lipooligo-
saccharide mutant background (42). HrpA also is essential for
N. meningitidis escape from the intracellular space after inva-
sion (46). FHA of B. pertussis is known to have multiple roles,
which include heparin binding, carbohydrate binding, CR3 and
adenylate cyclase interaction, and involvement in adherence,
invasion, proinflammatory response, and antiapoptotic charac-
teristics (1, 5, 19, 24, 25, 31, 32, 35, 38, 55). Therefore, it is
plausible that HrpA has multiple roles in N. meningitidis patho-
We recently showed that N. meningitidis could form biofilms
on transformed human bronchial epithelial (HBE) cells (34).
Our current work set out to determine if hrpA was regulated
and if HrpA was important in biofilm formation on HBE cells
in a flow cell system. Nonfimbrial adhesins are important for
mature biofilm formation in other organisms, including the
FhaBC system in Bordetella bronchiseptica (23). Our work
demonstrated the importance of HrpA in biofilm formation on
HBE cells in both wild-type and unencapsulated strains of N.
meningitidis. We further demonstrated the regulation of the
hrpA gene and the localization of the HrpA protein to the
MATERIALS AND METHODS
Strains and growth conditions. N. meningitidis strains NMB, NMB siaA-D
(ST4609; ST-8 complex, cluster A4), MC58 siaD (ST-32 ET-5), C311 siaD (un-
assigned multilocus sequence type), and their derivatives were used in this study
(see Table S1 in the supplemental material) (37, 45, 51). All strains contained the
Neisseria pGFP plasmid with the chloramphenicol cassette (11). Chloramphen-
* Corresponding author. Mailing address: Department of Microbi-
ology, 3-401 BSB University of Iowa, Iowa City, IA 52242. Phone:
(319) 335-7807. Fax: (319) 335-9006. E-mail: michael-apicella@uiowa
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 16 March 2009.
icol resistance has been reported to occur naturally (26, 44), and the use of this
as the selection is in accordance with NIH recombinant DNA guidelines (Section
IV-C-2-a). Strains were grown from frozen stock on GC agar (Becton Dickinson,
Sparks, MD) supplemented with 1% (vol/vol) IsoVitaleX. Kanamycin (75 ?g/ml)
and chloramphenicol (5 ?g/ml) were utilized as needed (Sigma, St. Louis, MO).
Escherichia coli strains were grown in Luria-Bertani (LB) broth (tryptone, 10
g/liter; yeast extract, 5 g/liter; NaCl, 10 g/liter) or on LB agar (LB broth with agar,
15 g/liter) (Becton Dickinson, Sparks, MD). Kanamycin (50 ?g/ml) and ampi-
cillin (125 ?g/ml) were utilized in E. coli cultures as needed (Sigma, St. Louis,
MO). Bacteria processed for protein or RNA were grown in RPMI 1640 with 1%
(vol/vol) IsoVitaleX (Gibco, Grand Island, NY).
Generation of mutants. Genomic DNA was isolated from strains NMB siaA-D
and MC58 siaD using ArchivePure DNA cell/tissue kits (5 Prime, Gaithersburg,
MD). Digoxigenin-labeled probes for Southern blottings were generated from
strain MC58 siaD using primers unique to regions of the five different hrpA-like
genes in that strain (see Table S1 in the supplemental material) (Roche, Mann-
heim, Germany). Five different Southern blottings were performed to determine
which of the five hrpA-like genes (NMB0493, NMB0497, NMB1214, NMB1768,
and NMB1779) were present in the NMB genome. A deletion mutant was made
of the putative hrpA gene in strain NMB by the replacement of the gene with a
kanamycin-sacB cassette from pJJ260 in both the encapsulated and unencapsu-
lated strain NMB backgrounds. The pJJ260 cassette has the P2 promoter from
Haemophilus influenzae driving the expression of the sacB cassette; however, the
P2 promoter is repressed by the tetR repressor. The mutation construct was
assembled in pBluescript SK?. The assembled construct was PCR amplified and
transformed into N. meningitidis strain NMB. Complementation was performed
by knock-in to replace the kan-sacB cassette with the original gene through the
transformation of genomic DNA from the wild-type organism and selection on
10% sucrose on LB agar with 1% IsoVitaleX and heat-inactivated chlortetracy-
cline (1 ?g/ml). Knock-in complementation was described previously for Neisse-
ria gonorrhoeae (43). Southern and Western blottings were performed to verify
the mutation, chromosomal complementation, and expected HrpA production
from these strains. All primers utilized in this study are in Table S1 in the
Real-time RT-PCR. RNA was isolated from N. meningitidis grown under the
conditions listed in Table 1 using the RNeasy kit (Qiagen, Valencia, CA). RNA
integrity was determined on an Agilent bioanalyzer (Agilent, Santa Clara, CA)
and used only if it had a relative integrity number above 8.0 on a scale of 1 to 10.
Bacterial samples were grown on three separate occasions in the appropriate
conditions, and RNA was isolated for use in real-time reverse transcriptase PCR
(RT-PCR). Primer/probe sets for genes hrpB, hrpA, and hrpS1were ordered from
Applied Biosystems (Austin, TX), and the primer probe set for the rmpM
transcript was ordered from Integrated DNA Technologies (Coralville, IA).
Standard curves were analyzed to determine the efficiency of amplification
and the ??CT method, with the efficiency correction as defined by Pfaffl, was
used for the analysis of the level (n-fold) of change (36). Normalized natural log
threshold cycle values of the control and test populations were analyzed by
two-sample t test for statistical significance. The efficiency of both primer sets was
the same, so no correction was needed for the two-sample t tests.
Polyclonal antibody development. The first 900 bp (positions 220 to 1119) after
the predicted encoding signal sequence of the hrpA gene was amplified and
cloned into pET15B (Novagen, EMD Biosciences, Madison, WI) and trans-
formed into BL21 pLysS chemically competent cells (Invitrogen, Carlsbad, CA).
Cultures were grown in 500 ml LB broth until an optical density (OD) of 0.3 at
600 nm was reached. The cultures were induced with 1 mM isopropyl-a ˆ-D-
thiogalactopyranoside (IPTG) and grown for three additional hours. The bacte-
ria were pelleted at 3,000 ? g for 15 min, and the pellet was resuspended in 50
ml 8 M urea, 20 mM sodium phosphate [pH 7.8], 500 mM NaCl buffer and
sonicated for three 20-s bursts. The lysed solution was spun at 3,000 ? g for 30
min, and the supernatant was filtered through a 0.2-?m filter. The filtered
material then was placed in a 50-ml GE superloop (GE Healthcare, Upsalla,
Sweden) on a GE fast-performance liquid chromatograph (GE Healthcare, Up-
sala Sweden) and chromatographed through a HisTrap Fast Flow nickel affinity
column (GE Healthcare, Upsalla, Sweden) at a rate of 2 ml per min, washed with
10 column volumes of 8 M urea buffer [pH 6.0], washed with another 10 column
volumes with 8 M urea buffer (pH 5.2), and finally eluted in 8 M urea buffer (pH
4.0). The eluted material then was concentrated to 0.5 ml in a Centricon YM10
filter at 3,000 ? g and dialyzed into 4 M urea. This material was passed through
a HiLoad 16/60 Superdex 75-pg gel filtration column in 4 M urea buffer to obtain
a single species of protein as visualized by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and continuous UV analysis of the fractionated material.
Stepwise dialysis in 2, 1, and 0.5 M urea was performed, and the resulting purified
protein was immunized into New Zealand White rabbits for polyclonal antibody
production, which was performed by the Iowa State University Hybridoma
Facility (Ames, IA).
Western blot analyses. Bacterial pellets were lysed in 4% (mol/vol) SDS in
H2O, and lysates were sheared through 23-gauge needles. The protein concen-
tration was determined by colorimetric assay (Pierce, Rockford, IL), and 5 ?g of
lysate was electrophoresed in each lane of NuPage 4 to 12% Bis-Tris polyacryl-
amide gels (Invitrogen, Carlsbad, CA). Proteins were transferred to an Immo-
bilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA) at 150
mA per gel for 16 h in Western Breeze transfer buffer (Invitrogen, Carlsbad, CA)
with 10% (vol/vol) methanol at 4°C. Membranes were blocked with Western
Breeze blocking agent (Invitrogen, Carlsbad, CA), anti-HrpA polyclonal anti-
body was applied at a 1:500 dilution, and goat anti-rabbit immunoglobulin G
horseradish peroxidase (HRP) secondary (Bio-Rad, Hercules, CA) was used at
1:10,000. The chemiluminescence of the HRP secondary conjugate was activated
with Pico-West super signal (Pierce, Rockford, IL) and developed on Kodak MR
film (Eastman Kodak, Rochester, NY). Mouse monoclonal antibody 2C3 against
the H.8 protein was used as a loading control with goat anti-mouse HRP sec-
ondary (Bio-Rad, Hercules, CA) at 1:10,000.
Tissue culture conditions. Simian virus 40-transformed HBE cells (ATCC
number CRL-9609) were cultured in minimal essential medium (MEM) tissue
culture medium with 10% (vol/vol) heat-inactivated fetal calf serum (FCS) and
2 mM L-glutamine at 37°C in the presence of 5% CO2(Gibco, Grand Island,
NY). HBE cells were trypsinized and placed onto 22- by 50-mm coverslips coated
with 0.5 mg/ml bovine collagen for use in biofilm chambers after the cells had
matured to at least 90% confluence as determined by visual examination.
Biofilms on HBE cells. N. meningitidis was cultured for 16 h as described above
and then suspended in 0.1? HBE tissue culture medium (MEM plus 10% FCS
and 2 mM L-glutamine) diluted in 1? phosphate-buffered saline (PBS), pH 7.4,
supplemented with IsoVitaleX (1:100, vol/vol) and 100 ?M sodium nitrite. Cul-
tures were vortexed for 15 s at high speed and then shaken at 150 rpm for 30 min
to disperse the bacteria (as verified by microscopic inspection) and adjusted to
1 ? 108organisms per ml (28). Bacterial suspensions were added to biofilm
chambers containing the HBE cell monolayers and allowed to incubate at 37°C
for 1 h before the initiation of medium flow. The flow rate was 150 ?l per min
TABLE 1. Results of real-time RT-PCR analyses of hrpB, hrpA, and hrpS1
RT-PCR condition hrpB hrpAhrpS1
P value of HrpA
Iron, 10 ?m
Desferol, 30 ?m
Serum, 5%, plus Desferol, 30 ?m
Anaerobic, 1 h
Anaerobic, 2 h
Anaerobic, 24 h
Biofilm, 48 h
Cell association, 5 h
?1.177 ? 0.19
1.238 ? 0.71
?1.458 ? 0.27
1.319 ? 0.34
4.479 ? 0.48
8.753 ? 0.69
4.539 ? 3.44
1.054 ? 0.71
14.445 ? 0.51
11.064 ? 3.43
?1.326 ? 0.69
1.319 ? 0.79
1.169 ? 0.43
?1.268 ? 1.07
3.968 ? 2.82
5.097 ? 2.18
2.840 ? 1.73
1.179 ? 0.84
11.344 ? 16.45
5.105 ? 4.89
?2.301 ? 0.12
?1.287 ? 0.22
?1.129 ? 0.19
?1.252 ? 0.06
2.437 ? 0.36
7.099 ? 0.73
3.001 ? 0.27
?1.084 ? 0.06
1.357 ? 0.65
2.885 ? 0.97
aData are the level of change (n-fold) from that of bacteria grown in aerobic broth conditions. The expression of the constitutive rmp gene was used to normalize
the hrpB, hrpA, and hrpS real-time RT-PCR results. P values from nonparametric two-sample t tests of the hrpA data are given (an asterisk indicates P ? 0.05).
2286NEIL AND APICELLAINFECT. IMMUN.
(14). Biofilms were allowed to mature for either 6 or 48 h before microscopy and
Biofilm apparatus. To assess biofilm growth on tissue culture cells, we
constructed a flowthrough chamber that permitted the insertion of a coverslip
coated with live HBE cells. The chamber was composed of three pieces. The
bottom was a 35- by 70- by 4-mm piece of acrylic with a 17-mm-wide, 45-mm
long, and 2-mm deep chamber machined out of the block. Medium inflow and
outflow ports were centered on the ends of the chamber at a 2-mm depth. A
1-mm-thick rubber gasket the size of the acrylic block with a 17- by 45-mm
section removed from the center was placed on top of the chamber bottom.
The top piece was acrylic, with the same 17- by 45-mm middle section
removed. The underside of the top piece has a 22- by 50-mm section ma-
chined out to the same depth as the coverslip to accommodate the tissue
culture cells. Ten screws held the apparatus together.
Confocal microscopy. Tissue culture cells were labeled with cell tracker
orange (Molecular Probes, Eugene, OR) prior to chamber inoculation. The
use of the Nikon Eclipse 80I confocal microscope, lasers, Comstat analysis,
and Volocity image rendering were described previously (29). We performed
five separate experiments for each growth condition and three separate Z-
series per flow cell.
Statistical analysis. For each biofilm condition, Z-series were taken on the
laser-scanning confocal microscope and analyzed by Comstat software for aver-
age height and total biomass (MathWorks, Natick, MA) (21). Comstat analysis is
a comprehensive digital analysis of a Z-series of images from a confocal micro-
scope. It allows the statistical analysis of the biomass and average height of the
biofilm across the entire Z-series of images. To accomplish this, each Z-series
photomicrograph is saved as a series of TIFF images that are converted into 8-bit
grayscale images in Comstat for pixel analysis (21). GraphPad Prism (San Diego,
CA) was used to calculate a nonparametric one-way analysis of variance
(ANOVA) with a Dunn’s multiple-comparison post test to compare biofilms and
generate graphs from a 12 to 18 Z-series. A P value of ?0.05 was considered
ELISA analysis. To determine the degree of piliation at the time of chamber
inoculation, we coated the enzyme-linked immunosorbent assay (ELISA) plates
with 100 ?l of an inoculum (OD at 600 nm of 0.1) and probed them with a rabbit
polyclonal antibody against PilE (a gift from Michael Jennings, University of
Queensland, Brisbane, Australia). The wild-type and complemented strains were
compared to the hrpA mutants as well as a pilE mutant of strain C311. Control
ELISA analysis against H8 with monoclonal antibody 2C3 was utilized to dem-
onstrate equal loading between strains.
HrpA localization. Biofilm inoculum bacteria were spun down and washed
with PBS and suspended to an OD of 0.3. A drop was placed on a microscope
slide and dispersed around a circle 1 cm in diameter and dried to completion.
The bacteria were blocked with 5% normal goat serum (Sigma, St. Louis, MO)
and labeled either with anti-HrpA antibody (1:100) and Cy5 conjugate secondary
antibody (1:250) (Jackson ImmunoResearch, West Grove, PA) or with the sec-
ondary antibody alone. Image analysis was performed on an Olympus BX-51
light microscope with fluorescent capabilities.
Strain NMB has paralogs to the MC58 genes NMB0497 and
NMB1779 as determined by Southern blot analysis (data not
shown). The cloning and sequencing of these regions demon-
strated that the NMB0497 paralog was identical to NMC0444
of the sequenced strain Fam18 (ST-11 complex/ET-37 com-
plex) and is referred to as hrpA for this study. Further analysis
demonstrated that the NMB1779 paralog was truncated from
the 5? end with the first 3,850 bp of the total 5,988 bp present
in the MC58 strain deleted. The remaining 3? fragment initi-
ated 557 bp downstream of the stop codon of the hrpA gene in
strain NMB with a 510-bp open reading frame located between
the two hrpA-related sequences. This truncated hrpA is re-
ferred to as hrpS1(NMC0446 homologue) for the duration.
Sequence analysis demonstrated greater than 99% sequence
identity from upstream of hrpB through downstream of the
hrpS3gene (NMC0450 homologue), corresponding to the
NMC0440-NMC0451 region of the Fam18 genome (GenBank
accession number FJ644951). A detailed view of this genetic
region in Fam18, Z2491, and MC58 was published previously
(49). A review of secretion systems in N. meningitidis com-
ments on the phenomenon of predicted pseudogenes or pre-
dicted noncoding regions that are 1,500 to 3,000 bp in length
with sequence similarities to the full-length hrpA genes (49).
These pseudogenes are scattered downstream of the predicted
hrpA gene in all currently sequenced genomes of N. meningi-
tidis. This phenomenon is postulated to be similar to the pilE
and pilS genetic loci in the Neisseria sp. genomes, in which the
pilS sequences are randomly incorporated into pilE by homol-
ogous recombination for the antigenic variation of the pilus
(49). We refer to these hrpA pseudogenes as hrpS regions to
maintain continuity to the pilE and pilS system.
Real-time RT-PCR was performed on RNA samples from
bacteria grown under conditions known to activate regulatory
pathways in N. meningitidis to determine if the hrpB, hrpA, or
hrpS1gene was regulated (Table 1) (15, 16, 18, 39). These
conditions were iron excess and iron depletion, the presence of
serum, anaerobic growth, biofilm growth, and cell association.
There was an upregulation of all three genes in anaerobic
conditions, between 2.4- to 8.7-fold depending on the time
spent in the anaerobic chamber. Interestingly, there was an
upregulation of all three transcripts after 5 h of cell association
in 24-well tissue culture plates. We also observed the upregu-
lation of bacteria detaching from biofilms on HBE cells in
which hrpB was upregulated 14.4-fold and hrpA was upregu-
lated 11.34-fold, while hrpS1was not upregulated. This is in-
teresting, considering that hrpBA are operonic and hrpS1ap-
pears to be operonic based on genetic structure (42). The lack
of the upregulation of the hrpS1transcript under this condition
may be explained by the size and stability of the large transcript
or by alternative terminator sites. Real-time RT-PCR results
of the hrpA transcript from anaerobic conditions (1 h, P ? 0.01;
2 h, P ? 0.0005) and cell association (P ? 0.025) were statis-
tically upregulated compared to levels for the hrpA transcript
of broth-grown organisms (Table 1). Significance was not
reached in the 24-h anaerobic conditions and the biofilm
planktonic conditions due to the large variation between sam-
ples. We are unsure of the reasons for this variation at this
time, but it could be due to instability of the mRNA.
Western blots demonstrated HrpA at both 220 and 180 kDa
in lysates of aerobically grown cultures (Fig. 1). This doublet
band was described previously in Western blots of HrpA (48).
Growth in anaerobic conditions consistently demonstrated a
significant decrease in the intensity of the 220-kDa band (Fig.
1A). This may indicate a more efficient processing of the 220-
kDa protein in low-oxygen conditions. When densitometry
measurements of the 180-kDa band for wild-type strains grown
aerobically or grown for 1, 2, or 24 h anaerobically in RPMI
medium were normalized to densitometry measurements of
the constitutively expressed H.8 band, statistically significant
increases in the 180-kDa band at 24 h (P ? 0.04) were observed
compared to results for normalized aerobic conditions (Fig.
1A). Densitometry was performed on Western blots from
three separate experiments. Strains MC58 siaA-D and C311
and the complemented NMB hrpA strain demonstrated the
production of HrpA antibody-reactive bands (Fig. 1B). The
MC58 strain also produced a third high-molecular-weight band
VOL. 77, 2009 N. MENINGITIDIS HrpA AND BIOFILM FORMATION2287
that was HrpA antibody reactive. This is not surprising, con-
sidering that MC58 has five hrpA paralogs.
We did not observe changes in the transcript of any gene in
the hrpBAS operon in biofilms collected at 48 h (Table 1).
However, an increase in the transcript of hrpBA after 5 h of cell
association as well as anaerobic conditions led us to determine
if HrpA had a role in biofilm formation on HBE cells. The
biofilm system allows for biofilm formation under conditions of
shear stress on HBE cells to mimic conditions in association
with human airway epithelial cells. First, growth curves with
NMB siaA-D, NMB siaA-D hrpA, and NMB siaA-D hrpA with
a chromosomal complementation of hrpA were performed,
and no growth defect was demonstrated in the mutant or
complemented strains (data not shown). We next screened the
strains used in biofilm analyses by ELISA against whole bac-
teria for the presence of pilus. All strains were equally piliated.
Images of 4?,6?-diamidino-2-phenylindole-stained HBE cells
and adherent bacteria were taken after the 1-h static incuba-
tion period and indicated that initial attachment was not sta-
tistically different for the wild type or hrpA mutant. There was
an average of 19 ? 7 bacteria per cell across all strains utilized
in this study at the time of flow initiation. Mutants of hrpA in
both the capsule-positive and capsule-negative strain of NMB
demonstrated no difference in average height and biomass at a
6-h time point (Fig. 2A and B). However, 48-h biofilms on
HBE cells demonstrated significantly reduced biofilm for-
mation in the hrpA mutants when analyzed for biomass
(encapsulated strains only) and average height (encapsu-
lated and unencapsulated strains) by confocal microscopy
and Comstat analysis (Fig. 2C and D). Images of the 6-h
biofilms can be seen in Fig. 3 and demonstrate that the
bacteria preferentially adhere to the HBE cells, as seen by
gaps in the HBE monolayer and a few bacteria filling these
gaps. The 48-h images (Fig. 4) demonstrate the growth of
the biofilm compared to that of the 6-h images and clearly
demonstrate the decrease in the biofilm in the hrpA mutant
compared to that of the wild type. In addition, Fig. 4 dem-
onstrates that the 48-h biofilm is affecting HBE cell conflu-
ence and cell morphology compared to the level of conflu-
ence of HBE cells at 6 h postinfection in Fig. 3. Uninfected
HBE monolayers incubated in flow cells for 48 h demon-
strated 80% survival and normal cell morphology. HBE cells
infected with strain NMB or NMB siaA-D had 60 and 70%
survival, respectively, at 48 h. The fact that there was 20%
cell death in the uninfected chambers indicates that the
chamber environment is not optimal for the growth of the
HBE cells, and the bacterial infection could exacerbate this
HrpA is a factor that is involved in escape from invaded
cells (46). We grew biofilms for 48 h on collagen-coated
FIG. 1. Western blots using a rabbit anti-HrpA antibody and
monoclonal antibody 2C3 that binds to the outer membrane protein
H.8. H.8 was used as the loading control for the individual samples
(A and B, lower images). (A) Lysates of strain NMB siaA-D grown
under aerobic conditions (lane 1) or anaerobic conditions for 1
(lane 2), 2 (lane 3), and 24 h (lane 4); lane 5 shows the growth of
NMB siaA-D hrpA. The normalized 180-kDa band in lane 4 was
significantly upregulated compared to the normalized 180-kDa
band in lane 1. (B) Lysates of strains MC58 siaD (lane 1), C311
(lane 2), NMB siaA-D (lane 3), NMB siaA-D hrpA, (lane 4), and a
lysate of NMB siaA-D hrpA complemented with hrpA (lane 5) grown
aerobically. Arrows indicate the HrpA-specific (220- and 180-kDa)
FIG. 2. Graphs of Comstat analyses of 6-h biomass (A) and aver-
age height (B), the same results for 48-h biomass (C), and the average
height of biofilms formed on HBE cells (D). A total of 12 to 15
Z-series were analyzed by ANOVA for the 6-h biofilm data, and 14 to
18 Z-series were analyzed for the 48-h biofilm data for each condition
tested from five to six different experiments. Data from the 6-h con-
ditions were not significant by ANOVA; however, data for both 48-h
biomass and average height were significant by ANOVA (P ? 0.0001).
Significance bars are results from ANOVA post tests.*, P ? 0.05;**,
P ? 0.01;***, P ? 0.0001. cap?, siaA-D capsular mutant; cap?comp,
NMB siaA-D hrpA with a chromosomal complementation of hrpA.
2288NEIL AND APICELLAINFECT. IMMUN.
coverslips to determine if the reason for reduced biofilm
formation on HBE cells at 48 h by the hrpA mutant was due
to an inability of the organism to escape from the cells and
then contribute to the biofilm structure. Biofilms of NMB
siaA-D and the NMB siaA-D hrpA complemented strain
formed significantly larger biofilms than the NMB siaA-D
hrpA mutant (Fig. 5). This suggests that egressing organisms
from the HBE cells do not make significant contributions to
the biofilm structure. Also, since the 6-h biofilms on HBE
cells were equivalent, this implies a role for HrpA in the
evolving structure of the biofilm and not necessarily as an
adhesin in our model system (42).
FIG. 3. Volocity-rendered confocal Z-series of 6-h biofilms on HBE cells infected with strains NMB (A), NMB hrpA (B), NMB siaA-D (C),
NMB siaA-D hrpA (D), and NMB siaA-D hrpA complemented with hrpA (E). Bacteria are green fluorescent protein labeled, and HBE cells are
labeled with cell tracker orange (red channel).
VOL. 77, 2009 N. MENINGITIDIS HrpA AND BIOFILM FORMATION2289
In contrast to previous publications, our Western blot
studies of strains NMB, MC58, and C311 failed to demon-
strate the secretion of HrpA into the supernatant of aero-
bically shaken broth cultures (42, 48). We performed fluo-
rescence microscopy to determine if the HrpA protein was
present on the surface of strain NMB. Figure 6 demon-
strates the presence of HrpA on the surface of the wild-type
NMB and the hrpA-complemented NMB siaA-D hrpA strain
but not the hrpA mutant or wild-type strain reacted with the
Cy5 conjugate secondary antibody alone.
FIG. 4. Volocity-rendered confocal Z-series of 48-h biofilms on HBE cells infected with strains NMB (A), NMB hrpA (B), NMB siaA-D (C),
NMB siaA-D hrpA (D), and NMB siaA-D hrpA complemented with hrpA (E). Bacteria are green fluorescent protein labeled, and HBE cells are
labeled with cell tracker orange (red channel).
2290 NEIL AND APICELLAINFECT. IMMUN.
Two-partner secretion systems support adherence to tissue
in B. pertussis, Moraxella catarrhalis, and enterotoxigenic E. coli
(2, 7, 13). The HrpAB two-partner secretion system of N.
meningitidis has a role in adherence to tissue culture cells but
only in the absence of capsule and with a lipooligosaccharide
truncation (42). HrpA also is important in escape from HeLa
cells (46). We used an expression-based approach to determine
if hrpA was regulated. If regulation occurred, we hoped that
this would point to a role for HrpA in N. meningitidis patho-
genesis. Culture after 5 h in the presence of HBE cells as well
as culture in anaerobic conditions resulted in the upregulation
of hrpA transcript. Interestingly, the significant upregulation of
transcript in anaerobic conditions at 1 and 2 h (Table 1) re-
sulted in a significant increase in HrpA protein only at 24 h of
anaerobic growth (for the 180-kDa band, P ? 0.04) compared
to that of organisms grown in aerobic conditions (Fig. 1). The
reason for this is not known at this time, but it could be due to
the inefficient transfer of HrpA onto the membrane for the
Western blots or other unknown reasons. The production of
the 180-kDa form of HrpA grown anaerobically is significantly
enhanced, based on the densitometry analysis of Western
blots, compared to that of the 180-kDa form during growth in
aerobic conditions. This may point to its function in the biofilm
process during conditions of low oxygen. Alternatively, it is
possible that a sequence-specific protease is more active in the
anaerobic conditions and cleaves HrpA to the 180-kDa form.
The results of the real-time RT-PCR experiments directed us
to evaluate the role of HrpA in biofilm formation. A significant
reduction in biofilm biomass and average height for hrpA mutants
was demonstrated in the 48-h biofilms but not in the 6-h biofilms.
There are three possible explanations for the hrpA mutant phe-
notype. The initial finding that HrpA is an adhesin in an unen-
capsulated lipooligosaccharide truncation strain of N. meningitidis
supports the first possibility, that biofilms are reduced because of
FIG. 5. Comstat analyses of 48-h biofilms grown on collagen-
coated coverslips in the same flow cells and media as biofilms produced
on HBE cells. A total of 10 Z-series were analyzed from a minimum of
four experiments for each condition. ?, P ? 0.05; ??, P ? 0.01; ???, P ?
FIG. 6. Fluorescence microscopy of whole bacteria immunolabeled with anti-HrpA with a Cy5 secondary antibody. (A) NMB siaA-D; (B) NMB
siaA-D with secondary antibody alone; (C) NMB siaA-D hrpA; and (D) NMB siaA-D hrpA complemented with hrpA. Scale bar, 10 ?m.
VOL. 77, 2009 N. MENINGITIDIS HrpA AND BIOFILM FORMATION2291
decreased adherence to HBE cells. If fewer numbers of initial
bacteria bound to the HBE cells, this would result in fewer bac-
development. However, as stated above, the levels of bacterial
adherence at the time of the initiation of flow were comparable
for all strains tested. Furthermore, biofilm formation at the 6-h
time point was equivalent among all strains based on Comstat
analyses (Fig. 2A and B). This suggests that adherence to the
HBE cells was not a factor in the differences in biofilm develop-
ment between the hrpA mutant and the wild type. The second
escape from invaded tissue (46). If only wild-type bacteria can
escape the tissue culture cells, then the wild-type organisms could
provide an additional population of bacteria to contribute to the
biofilm after escape while the hrpA mutant would be deficient.
The third possibility is that HrpA has a role in the maintenance of
the biofilm structure. The 48-h biofilms grown on collagen dem-
onstrated that HrpA was involved in biofilm development be-
cause of the biofilm defect in the absence of cells (Fig. 5). FHA
previously was implicated in multiple functions in B. pertussis,
including binding to self and host ligands (31, 35, 55). This exam-
ple of multiple roles for FHA demonstrates the possibility of
HrpA having multiple domains for interaction with different cel-
lular or bacterial ligands. It also is possible that the different
processed forms of HrpA have different roles.
Based on our results, we hypothesize that HrpA is active
mainly in its 180-kDa processed form and its presence is en-
hanced in anaerobic environments, as suggested by the real-
time RT-PCR data, which demonstrate the upregulation of the
hrpA transcript (Table 1), and the Western blot data, which
demonstrates the enhanced processing of HrpA (Fig. 1A). This
corresponds to the data of a biofilm defect in the HrpA mu-
tant. Biofilms are known to develop oxygen gradients as they
grow (54). This suggests that microaerophilic or anaerobic
environments develop within larger biofilms, where the hrpA
transcript could be upregulated and the production of the
180-kDa form of HrpA could be enhanced. Our data suggest
that HrpA has a role in maintaining the biofilm structure,
possibly through bacteria-bacteria interaction. We hypothesize
that mutant meningococci lacking HrpA form smaller biofilms
with smaller oxygen gradients, since the anaerobically active
form of the protein is absent in the mutant strain and therefore
not able to hold the larger biofilm together.
We are unsure of the reason why HrpA remains surface
bound in our study, while studies from other groups found
HrpA in the supernatant (42, 48). It may be because the pre-
vious studies grew their cultures to ODs that most likely rep-
resented stationary-phase organisms, while ours were still in
the log phase of growth. Since Neisseria organisms bleb and
undergo autolysis, these studies could be detecting HrpA from
lysed organisms or HrpA bound to membrane fragments that
are too small to pellet in the low-speed centrifugation that is
used to pellet the bacteria (33, 40). Alternatively, N. meningi-
tidis may actively release bound HrpA at stationary phase, and
we cannot exclude the possibility that our strains are unable to
release HrpA from the surface. One candidate to aid in the
release of HrpA from the meningococcal surface is NalP. This
protein has 30% homology to the SphB1 protein that is in-
volved in the final maturation of FHA in B. pertussis (9). The
nalP gene is likely phase variable in N. meningitidis due to a
stretch of ?10 cytosine residues in the coding sequence that
participate in slip-stranded mispairing to turn the gene on and
off (47, 50). However, the sequence analysis of strain NMB
demonstrated nalP is phase on (data not shown).
Studies of the two-partner secretion system of N. meningiti-
dis have led to interesting discoveries regarding the pathogen-
esis of epithelial cell layer infection models. The first is that
HrpA is important in intracellular escape (46). The mechanism
by which this occurs is not understood. The second is that
HrpA has a role in biofilm formation on epithelia, as demon-
strated in this study. Biofilms may be important for N. menin-
gitidis during nasopharyngeal colonization. The genes encoding
the HrpA proteins are found in virtually all strains of N. men-
ingitidis (42, 48). Since the HrpA paralog FHA is a component
of the acellular B. pertussis vaccine, future research should
determine if HrpA undergoes antigenic variation, which could
limit the usefulness of HrpA in a broadly reactive meningo-
coccal vaccine (12). In addition, investigations of the cellular or
self receptors accounting for HrpA protein involvement in
bacteria-cell or bacteria-bacteria interactions should be under-
This work was supported by AI045728 and AI007511.
We thank Yu-Hui Chang for assistance in statistical analysis.
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Editor: J. N. Weiser
VOL. 77, 2009N. MENINGITIDIS HrpA AND BIOFILM FORMATION 2293