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. Greiner, L. L., J. L. Edwards, J. Shao, C. Rabinak, D. Entz, and M. A.
Apicella. 2005. Biofilm formation by Neisseria gonorrhoeae. Infect. Immun.
15. Grifantini, R., E. Bartolini, A. Muzzi, M. Draghi, E. Frigimelica, J. Berger,
F. Randazzo, and G. Grandi. 2002. Gene expression profile in Neisseria
meningitidis and Neisseria lactamica upon host-cell contact: from basic re-
search to vaccine development. Ann. N. Y. Acad. Sci. 975:202–216.
16. Grifantini, R., S. Sebastian, E. Frigimelica, M. Draghi, E. Bartolini, A.
Muzzi, R. Rappuoli, G. Grandi, and C. A. Genco. 2003. Identification of
iron-activated and -repressed Fur-dependent genes by transcriptome analysis
of Neisseria meningitidis group B. Proc. Natl. Acad. Sci. USA 100:9542–9547.
17. Gue ´din, S., E. Willery, J. Tommassen, E. Fort, H. Drobecq, C. Locht, and F.
Jacob-Dubuisson. 2000. Novel topological features of FhaC, the outer mem-
brane transporter involved in the secretion of the Bordetella pertussis fila-
mentous hemagglutinin. J. Biol. Chem. 275:30202–30210.
18. Hagen, T. A., and C. N. Cornelissen. 2006. Neisseria gonorrhoeae requires
expression of TonB and the putative transporter TdfF to replicate within
cervical epithelial cells. Mol. Microbiol. 62:1144–1157.
19. Hannah, J. H., M. F. Renauld, G. Locht, and C. M. J. Brennan. 1994.
Sulfated glycoconjugate receptors for the Bordetella pertussis adhesin fila-
mentous hemagglutinin (FHA) and mapping of the heparin-binding domain
on FHA. Infect. Immun. 62:5010–5019.
20. Henderson, I. R., F. Navarro-Garcia, M. Desvaux, R. C. Fernandez, and D.
Ala’Aldeen. 2004. Type V protein secretion pathway: the autotransporter
story. Microbiol. Mol. Biol. Rev. 68:692–744.
21. Heydorn, A., A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K.
Ersboll, and S. Molin. 2000. Quantification of biofilm structures by the novel
computer program COMSTAT. Microbiology 146:2395–2407.
22. Hodak, H., B. Clantin, E. Willery, V. Villeret, C. Locht, and F. Jacob-
Dubuisson. 2006. Secretion signal of the filamentous haemagglutinin, a
model two-partner secretion substrate. Mol. Microbiol. 61:368–382.
23. Irie, Y., S. Mattoo, and M. H. Yuk. 2004. The Bvg virulence control system
regulates biofilm formation in Bordetella bronchiseptica. J. Bacteriol. 186:
24. Ishibashi, Y., D. A. Relman, and A. Nishikawa. 2001. Invasion of human
respiratory epithelial cells by Bordetella pertussis: possible role for a filamen-
tous hemagglutinin Arg-Gly-Asp sequence and ?5?1 integrin. Microb.
25. Ishibashi, Y., K. Yoshimura, A. Nishikawa, S. Claus, C. Laudanna, and D. A.
Relman. 2002. Role of phosphatidylinositol 3-kinase in the binding of Bor-
detella pertussis to human monocytes. Cell Microbiol. 4:825–833.
26. Jorgensen, J., S. Crawford, and K. Fiebelkorn. 2005. Susceptibility of Neis-
seria meningitidis to 16 antimicrobial agents and characterization of resis-
tance mechanisms affecting some agents. J. Clin. Microbiol. 43:3162–3171.
27. Kajava, A. V., and A. C. Steven. 2006. The turn of the screw: variations of the
abundant beta-solenoid motif in passenger domains of Type V secretory
proteins. J. Struct. Biol. 155:306–315.
28. Lappann, M., J. A. J. Haagensen, H. Claus, U. Vogel, and S. Molin. 2006.
Meningococcal biofilm formation: structure, development and phenotypes in
a standardized continuous flow system. Mol. Microbiol. 62:1292–1309.
29. Lim, K. H., C. E. Jones, R. N. van den Hoven, J. L. Edwards, M. L. Falsetta,
M. A. Apicella, M. P. Jennings, and A. G. McEwan. 2008. Metal binding
specificity of the MntABC permease of Neisseria gonorrhoeae and its influ-
ence on bacterial growth and interaction with cervical epithelial cells. Infect.
30. Mazar, J., and P. A. Cotter. 2006. Topology and maturation of filamentous
haemagglutinin suggest a new model for two-partner secretion. Mol. Micro-
31. Menozzi, F. D., P. E. Boucher, G. Riveau, C. Gantiez, and C. Locht. 1994.
Surface-associated filamentous hemagglutinin induces autoagglutination of
Bordetella pertussis. Infect. Immun. 62:4261–4269.
32. Mobberley-Schuman, P. S., and A. A. Weiss. 2005. Influence of CR3
(CD11b/CD18) expression on phagocytosis of Bordetella pertussis by human
neutrophils. Infect. Immun. 73:7317–7323.
33. Namork, E., and P. Brandtzaeg. 2002. Fatal meningococcal septicaemia with
“blebbing” meningococcus. Lancet 360:1741.
34. Neil, R. B., J. Shao, and M. A. Apicella. 2009. Biofilm formation on human
airway epithelia by encapsulated Neisseria meningitidis serogroup B. Mi-
crobes Infect. 11:281–287.
35. Perez Vidakovics, M. L., Y. Lamberti, W. L. van der Pol, O. Yantorno, and
M. E. Rodriguez. 2006. Adenylate cyclase influences filamentous haemag-
glutinin-mediated attachment of Bordetella pertussis to epithelial alveolar
cells. FEMS Immunol. Med. Microbiol. 48:140–147.
36. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in
real-time RT-PCR. Nucleic Acids Res. 29:e45.
37. Post, D. M., M. R. Ketterer, N. J. Phillips, B. W. Gibson, and M. A. Apicella.
2003. The msbB mutant of Neisseria meningitidis strain NMB has a defect in
lipooligosaccharide assembly and transport to the outer membrane. Infect.
38. Prasad, S. M., Y. Yin, E. Rodzinski, E. I. Tuomanen, and H. R. Masure.
1993. Identification of a carbohydrate recognition domain in filamentous
hemagglutinin from Bordetella pertussis. Infect. Immun. 61:2780–2785.
39. Rock, J. D., M. R. Mahnane, M. F. Anjum, J. G. Shaw, R. C. Read, and J. W.
Moir. 2005. The pathogen Neisseria meningitidis requires oxygen, but sup-
plements growth by denitrification. Nitrite, nitric oxide and oxygen control
respiratory flux at genetic and metabolic levels. Mol. Microbiol. 58:800–809.
40. Rosenstein, N. E., B. A. Perkins, D. S. Stephens, T. Popovic, and J. M.
Hughes. 2001. Meningococcal disease. N. Engl. J. Med. 344:1378–1388.
41. Scarselli, M., D. Serruto, P. Montanari, B. Capecchi, J. Adu-Bobie, D. Veggi,
R. Rappuoli, M. Pizza, and B. Arico. 2006. Neisseria meningitidis NhhA is a
multifunctional trimeric autotransporter adhesin. Mol. Microbiol. 61:631–
42. Schmitt, C., D. Turner, M. Boesl, M. Abele, M. Frosch, and O. Kurzai. 2007.
A functional two-partner secretion system contributes to adhesion of Neis-
seria meningitidis to epithelial cells. J. Bacteriol. 189:7968–7976.
43. Seib, K. L., H. J. Wu, Y. N. Srikhanta, J. L. Edwards, M. L. Falsetta, A. J.
Hamilton, T. L. Maguire, S. M. Grimmond, M. A. Apicella, A. G. McEwan,
and M. P. Jennings. 2007. Characterization of the OxyR regulon of Neisseria
gonorrhoeae. Mol. Microbiol. 63:54–68.
44. Shultz, T., J. Tapsall, P. White, C. Ryan, D. Lyras, J. Rood, E. Binotto, and
C. Richardson. 2003. Chloramphenicol-resistant Neisseria meningitidis con-
taining catP isolated in Australia. J. Antimicrob. Chemother. 52:856–859.
45. Stabler, R. A., G. L. Marsden, A. A. Witney, Y. Li, S. D. Bentley, C. M. Tang,
and J. Hinds. 2005. Identification of pathogen-specific genes through mi-
croarray analysis of pathogenic and commensal Neisseria species. Microbi-
46. Tala, A., C. Progida, M. De Stefano, L. Cogli, M. R. Spinosa, C. Bucci, and
P. Alifano. 2008. The HrpB-HrpA two-partner secretion system is essential
for intracellular survival of Neisseria meningitidis. Cell Microbiol. 10:2461–
47. Turner, D. P., K. G. Wooldridge, and D. A. Ala’Aldeen. 2002. Autotrans-
ported serine protease A of Neisseria meningitidis: an immunogenic, surface-
exposed outer membrane, and secreted protein. Infect. Immun. 70:4447–
48. van Ulsen, P., L. Rutten, M. Feller, J. Tommassen, and A. van der Ende.
2008. Two-partner secretion systems of Neisseria meningitidis associated with
invasive clonal complexes. Infect. Immun. 76:4649–4658.
49. van Ulsen, P., and J. Tommassen. 2006. Protein secretion and secreted
proteins in pathogenic Neisseriaceae. FEMS Microbiol. Rev. 30:292–319.
50. van Ulsen, P., L. van Alphen, J. ten Hove, F. Fransen, P. van der Ley, and
J. Tommassen. 2003. A neisserial autotransporter NalP modulating the pro-
cessing of other autotransporters. Mol. Microbiol. 50:1017–1030.
51. Virji, M., K. Makepeace, I. R. Peak, D. J. Ferguson, M. P. Jennings, and
E. R. Moxon. 1995. Opc- and pilus-dependent interactions of meningococci
with human endothelial cells: molecular mechanisms and modulation by
surface polysaccharides. Mol. Microbiol. 18:741–754.
52. Virji, M., M. K., Ferguson, D. J., Achtman, M., and E. R. Moxon. 1993.
Meningococcal Opa and Opc proteins: their role in colonization and inva-
sion of human epithelial and endothelial cells. Mol. Microbiol. 10:499–510.
53. Yasukawa, K., P. Martin, C. R. Tinsley, and X. Nassif. 2006. Pilus-mediated
adhesion of Neisseria meningitidis is negatively controlled by the pilus-retrac-
tion machinery. Mol. Microbiol. 59:579–589.
54. Yoon, S. S., R. F. Hennigan, G. M. Hilliard, U. A. Ochsner, K. Parvatiyar,
M. C. Kamani, H. L. Allen, T. R. DeKievit, P. R. Gardner, U. Schwab, J. J.
Rowe, B. H. Iglewski, T. R. McDermott, R. P. Mason, D. J. Wozniak, R. E.
Hancock, M. R. Parsek, T. L. Noah, R. C. Boucher, and D. J. Hassett. 2002.
Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to
cystic fibrosis pathogenesis. Dev. Cell 3:593–603.
55. Zaretzky, F. R., M. C. Gray, and E. L. Hewlett. 2002. Mechanism of associ-
ation of adenylate cyclase toxin with the surface of Bordetella pertussis: a role
for toxin-filamentous haemagglutinin interaction. Mol. Microbiol. 45:1589–
Editor: J. N. Weiser
VOL. 77, 2009N. MENINGITIDIS HrpA AND BIOFILM FORMATION 2293