Meningococcal adhesion suppresses pro-apoptotic gene expression and
promotes expression of genes supporting early embryonic and cytoprotective
signaling of human endothelial cells.
Irena Linhartova1, Marek Basler1, Jeffrey Ichikawa2, Vladimir Pelicic3, Radim Osicka1,
Stephen Lory2, Xavier Nassif3 and Peter Sebo1
1Laboratory
of Molecular Biology of Bacterial Pathogens, Institute of Microbiology,
Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic
2Department
of Microbiology and Molecular Genetics, Harvard Medical School,
Warren Alpert Building, Rm 363, 200 Longwood Avenue, Boston, MA 02115
3INSERM
U570, Faculté de Médecine René Descartes, 156 rue de Vaugirard, 75015
Paris, France.
Running title: Human gene expression influenced by meningococcal adhesion
Keywords: Neisseria meningitidis, adherence, signaling, microarray
*For correspondence.
Dr. Irena Linhartova
Laboratory of Molecular Biology of Bacterial Pathogens
Czech Academy of Sciences
Institute of Microbiology
Videnska 1083; 142 20 Prague 4; Czech Republic
Tel: (+420) 241 062 762
Fax: (+420) 241 062 152
E-mail: linhart@biomed.cas.cz
1
Abstract:
Neisseria meningitidis colonizes human nasopharynx and occasionally causes
lethal or damaging septicemia and meningitis. Here we examined the adherencemediated signaling of meningococci to human cells by comparing gene expression
profiles of human umbilical vein endothelial cells (HUVEC) infected by adherent wildtype, frpC-deficient mutant, or the non-adherent (ΔpilD) N. meningitidis. Pili-mediated
adhesion of meningococci resulted in alterations of expression levels of human
genes known to regulate apoptosis, cell proliferation, inflammatory response,
adhesion and genes for signaling pathway proteins such as TGF-β/Smad, Wnt/βcatenin, and Notch/Jagged. This reveals that adhering piliated meningocci
manipulate host signaling pathways controlling cell proliferation while establishing a
commensal relationship.
2
Introduction
The Gram-negative bacterium Neisseria meningitidis is an obligately human
commensal and at the same time it belongs to leading agents of sepsis and
meningitis. Meningococcal infection starts by colonization of the nasopharyngeal and
tonsillar mucosa, where the bacteria adhere by an active process. Microbial entry into
the epitelium requires signaling from the invading pathogen to the host cell (Wilson,
et al., 1996, Finlay & Cossart, 1997, Nakagawa, et al., 2004) and adhesion to human
cells through type IV pili (Nassif, et al., 1994, Rudel, et al., 1995).
Bacteria and viruses both utilize and manipulate infected cells and employ a
variety of mechanisms to modulate innate cellular defense mechanism. For example,
prevention of host cell apoptosis may represent a survival strategy in chronic
infections by certain bacteria, such as Porphyromonas gingivalis (Nakhjiri, et al.,
2001), Chlamydia pneumoniae (Fischer, et al., 2001) or Helicobacter pylori (Shirin, et
al., 2000). For Neisseria species, infection of human urethral epithelium with
Neisseria gonorrhoeae was shown to yield upregulation of several host anti-apoptotic
factors (Binnicker, et al., 2003, Binnicker, et al., 2004). Similar results were recently
obtained by analysis of expression of selected genes in human meningeal-derived
cells, treated with meningococcal secreted proteins or infected with live meningococci
(Robinson, et al., 2004).
Bacterial toxins are key factors of virulence during infection and often
modulate the most essential cellular processes. In contrast to a number of Gramnegative bacterial pathogens, however, no proteinaceous exotoxins have yet been
implicated in the pathogenesis of invasive meningococcal disease. In 1993,
Thompson and colleagues (Thompson, et al., 1993, Thompson, et al., 1993)
discovered in meningococci two homologous secreted proteins, FrpC and FrpC-like
(initially called FrpA), that belong to the repeat in toxin (RTX) family. A number of
RTX proteins was already shown to be involved in virulence of other Gram-negative
genera, such as Actinobacillus, Bordetella, Escherichia, Moraxella, Morganella,
Pasteurella, Proteus, and Vibrio (Lally, et al., 1999, Welch, 2001). In a previous
study, we have shown that genes encoding FrpC-like proteins of variable size are
present in most of the clinical isolates of N. meningitidis and that sera of most
patients who underwent invasive meningococcal disease contain antibodies
specifically recognizing these proteins (Osicka, et al., 2001). The biological activity of
FrpC-like proteins however remains unknown (Forman, et al., 2003).
3
Here we aimed to explore the specific effects of pilus-mediated adhesion and
RTX-protein production on gene expression levels of HUVEC cells infected by
meningococci.
4
Materials and Methods
Bacterial strains and growth conditions.
N. meningitidis strain MC58 was previosuly described (McGuinness, et al., 1991), as
well as the following isogenic derivatives, pilD insertion-deletion mutant (MC58ΔpilD)
(Carbonnelle, et al., 2005) and frpC-/frpC-like- insertion-deletion mutant
(MC58ΔfrpC/frpC-like) (Forman, et al., 2003). Bacteria from frozen stocks were
grown overnight at 37°C in atmosphere containing 5% CO2 on GCB agar (Difco)
plates with Kellogg’s supplement (Kellogg, et al., 1963) modified as described
elsewhere (Pelicic, et al., 2000). Prior to infection of HUVEC cells, meningococci
were washed, resuspended in RPMI 1640 supplemented with glutamax I (GibcoBRL) and 10% heat-inactivated fetal calf serum (infection medium) to OD600=0.05
and grown for 2 hours at 37°C prior use for infection of HUVEC cells.
Culture of primary human umbilical vein endothelial cells (HUVECs)
HUVEC cells (PromoCell, Heidelberg, Germany) were used between passages 2 and
10 and were grown in 150 cm2 tissue-culture flasks in Human Endothelial Medium
(GIBCO-Invitrogen Corporation) supplemented with 10% heat-inactivated fetal calf
serum (FCS), 1 ng/ml β-FGF (Boehringer-Mannheim, Meylan, France), 2 mM Lglutamine (Life Technologies, Grand Island), 0.5 UI/ml heparin (Sigma, Saint Louis)
and 1.25 mg/ml endothelial cell growth supplement (Sigma, Saint Louis). Cells were
seeded at 8 x 103 cells/cm2 and grown at 37°C in a humidified 5% CO2 atmosphere
for 3–4 days. Confluent cultures were used for all experiments.
Infection of HUVEC cells with adhering bacteria.
HUVEC cells were grown to confluence (approximately 3 x 104 cells/cm2) in 150 cm2
flasks in HUVEC culture medium. Three hours prior to infection by N. meningitidis cell
cultures were incubated with infection medium. The initial multiplicity of infection was
10. Cell monolayers infected with adherent wild-type or ΔfrpC strains were washed
every hours for 1, 4, or 6 hours with prewarmed fresh infection medium. This allowes
the bacteria to initially adhere for one hour, further increase in the number of bacteria
adhering with the monolayers was the consequence of a bacterial multiplicaton on
the apical side of the monolayer. Monolayers infected with non adherent pilD strains
5
were not subjected to washing. Three independent experiments were performed for
the 4 hours time points and two experiments for the 1 and 6 hours time points.
Adherence
Adhesion assays were performed in 24-well plates, using confluent HUVEC cultures
and MOI of 200. Incubation was carried out for up to 5 h, the medium was replaced
and cells were washed every hour to minimise monolayer reinfection by free-floating
bacteria. Adherent bacteria were counted.
RNA isolation
Following infection, total RNA was extracted from infected and uninfected cells using
Trizol reagent (Invitrogen Life Technologies) according to the manufacturer’s
protocol. Total cell RNA (totRNA) was purified on RNeasy minicolumns (Qiagen) and
by NaOAc/ethanol precipitation. Purified totRNA was transcribed into cDNA
according to the Affymetrix protocol. Briefly: 40 g of totRNA was reverse-transcribed
into single-stranded cDNA in a reaction mixture containing 5 μl (200 U/ μl)
SuperScript II reverse transcriptase (Invitrogen Life Technologies), 1 μl (100 pmol/ μl)
T7-(dT)24 primer (Generi Biotech) and each deoxynucleoside triphosphate (1 μl dNTP
mix 10mmol/ml)(Invitrogen Life Technologies). The reaction mixture for second
strand synthesis contained E.coli DNA ligase (1 μl, 10 U/ μl), E.coli DNA polymerase
I (4 μl, 10 U/ μl), E.coli RNase H (1 μl, 2 U/ μl), T4 DNA polymerase (2 μl, 5 U/ μl)
and each deoxynucleoside triphosphate (3 μl , 10 mM) (Invitrogen Life
Technologies). Resulting cDNA samples were purified by
phenol/chloroform/isoamylalcohol extraction and NH4OAc/ethanol precipitation.
Microarray analysis
cRNA Target Synthesis and GeneChip Hybridization
cDNA was used for the in vitro transcription reaction performed by the MEGA Script
T7 in vitro transcription kit (Ambion, Austin, TX, USA), including biotinylated
ribonucleotides in the reaction mixture (biotinyl-11-CTP and biotinyl-16-UTP, Enzo
New York, NY, USA). After purification of the in vitro transcription reaction products
(RNeasy; Qiagen), 15 µg of the biotinylated cRNA were heat-fragmented (95 °C, 35
min) and hybridized overnight to the human HG-U133A oligonucleotide microarrays
(Affymetrix, Santa Clara, CA, USA) at 45 °C in a rotary oven. The final washing,
6
staining and scanning steps were performed using the Affymetrix GeneChip fluidic
station and Agilent GeneArray scanner (Agilent, Palo Alto, CA, USA), following
manufacturer’s instructions.
Data Analysis
Cell Intensity Files (CEL) were analyzed using the Robust Multi-array Analysis
algorithm implemented in the BioConductor package. Data were normalized using
the quantile method and gene expression signal calculation was based on the
Perfect Match values from each probe set, as described previously (Irizarry, et al.,
2003). All data, including CEL files, are stored in MIAME compliant database Gene
Expression Omnibus at the National Center for Biotechnology Information (Barrett, et
al., 2005).
The p-value below 0.05 and the ratio over 1.7 times were used as thresholds
for inclusion into the list of differentially expressed genes. Genes flagged as Absent
by MAS 5.0 algorithm in both compared experimental groups were excluded.
7
Results and Discussion
Adherence-independent effects of meningococcal infection on gene
expression in HUVEC cells
Prior to analyzing the specific effects of adherence of meningococci, we first sought
to identify alterations in HUVEC gene expression that were independent of bacterial
adhesion and were due to presence of meningococcal lipopooligosaccharide, activity
of secreted neisserial proteins and/or due to hypoxia caused by oxygen consumption
by the growing bacteria. Towards this aim, we compared transcriptomes of noninfected HUVECs incubated in sterile medium, to the transcriptomes of HUVECs
infected at an MOI=10 by either the adherent wild type N. meningitidis MC58, or its
non-adherent ΔpilD mutant, unable to produce type IV pili (Nunn & Lory, 1991). In the
first hour we did not find any changes in expressed genes.
As shown in Table I, in comparison to non-infected control cells, a large number of
genes exhibited an altered expression in time point 4 and 6 in cells infected by either
of the two types of meningococcal strains, independently of whether the bacteria
produced pili and adhered to cells or not. Indeed, upregulation of many of these
genes, responding to bacterial presence in general, was previously observed in cells
exposed to other Gram-negative pathogenic bacteria and/or to meningococci (Dixon,
et al., 1999, Wells, et al., 2001, Zhao, et al., 2001, Christodoulides, et al., 2002).
These genes could be grouped into several classes, such as genes involved in
apoptosis, stress response, hypoxia, cytoskeletal protein reorganization, cell
adhesion and cytokine, chemokine and other inflammatory mediator production
(Table 1; All data including Affymetrix .CEL files are deposited in NCBI Gene
Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible
through GEO series accession number GSE4646).
In particular, strong induction of genes involved in inflammatory processes,
cell adhesion and motility was observed in infected HUVEC cells, such as
upregulation of genes for IL-8 and monocyte chemotactic protein-1 (MCP-1). This is
consistent with elevated levels of IL-8 and MCP-1 in the cerebrospinal fluid of
patients with bacterial meningitis (Wells, et al., 2001). Infection by both piliated and
non-piliated meningococci resulted also in upregulation of transcription of genes for
chemokines with inflammatory and growth-regulatory properties, such as CXCL1,
CXCL2, CXCL3, the endothelial adhesion molecule 1 selectin E (SELE) and
8
adhesion molecules ICAM-1 and VCAM-1. Also noteworthy is the observed strong
upregulation of the gene encoding cyclooxygenase-2 (COX-2), the tumor necrosis
factor alpha-induced protein 2 (TNFAIP2), superoxide dismutase 2 (SOD2, MnSOD)
and IL-6.
On the other hand, also genes involved in protection against excessive
inflammatory processes were found to be upregulated in infected HUVECs, such as
the gene for zinc finger protein A20 (TNFAIP3) (Lee, et al., 2000) and the A20binding inhibitor of NF-κB activation ABIN1 (TNIP1 or NAF1) (Heyninck, et al., 2003).
Altogether, these results are consistent with previous observations pointing to
an active participation of endothelial cells in immune response against Gramnegative bacteria. Endothelial cells appear, indeed, to be one of the first cell types
responding to LPS of Gram-negative bacteria when this is liberated into circulation.
Moreover, infection by N. meningitidis resulted in more than 10-fold enhanced
transcription of the gene for CD69, which is a marker of activated leukocytes and
lymphocytes and was not reported previously to be expressed by other cell types.
Efect of FrpC/FrpA proteins on gene expresion in HUVEC cells
To examine the role of the two homologous and secreted RTX proteins (FrpC and
FrpC-like), we next specifically compared expression profiles of cells infected by
either the wild-type N. meningitidis MC58 or its ΔfrpC/frpC-like double mutant, unable
to produce any of the two proteins (Forman, et al., 2003). Independently of
production FrpC and FrpC-like proteins by meningococci (data not shown) no
statistically significant difference was observed between the transcriptomes of
HUVECs infected by the two different strains, as documented at NCBI Gene
Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) GEO Series accession
number GSE4646. Hence, this experiment failed to yield any hints on the potential
activity of FrpC proteins on human endothelial cells, despite of infection times as long
as 6 hours and despite of the proteins being naturally produced and immunogenic in
meningococcal infections in humans. It should be pointed out that this negative result
demonstrated the reproducibility of the performed experiments and the robustness of
the data obtained.
Specific alterations of HUVEC transcriptome due to adherent growth of
meningococcal microcolonies on HUVEC cell surface
9
Type IV pili-mediated adherence of meningococci was previously shown to yield
growth of meningococcal microcolonies on HUVEC cell surface under similar
experimental conditions, as those used here for infection of HUVECs by the various
strains (Pujol, et al., 1999). To unravel the specific transcriptome alterations caused
by signaling of adherent meningococci growing on cell surface, we subtracted all
gene expression alterations that were common to transcriptomes of HUVECs
infected by both adherent and non-adherent bacteria, as compared to the
transcriptome of uninfected cells. As summarized in Table 2, indeed, numerous
genes were found that exhibited expression levels specifically and significantly
altered only in HUVECs infected by the two adherent strains, the wild-type and the
ΔfrpC/frpC-like double mutant bacteria. These genes could be grouped into several
functional categories and comprised, in particular, cell proliferation, differentiation,
apoptosis, cell adhesion and modulation of the inflammatory and immune response.
The data discussed here have been deposited in NCBIs Gene Expression Omnibus
(GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series
accession number GSE4646.
Specific up-regulation was observed for genes involved in three important
early embryonic signaling pathways: the TGF-β/Smad pathway, Notch - Delta/Jagged
pathway and the Wnt/β-catenin pathway, respectively. Enhanced transcription was
detected for genes of several other transcription factors belonging to various protein
families, such as forkhead/winged helix family, Ets and MAF family. Up-regulation
was also observed for kelch protein family, which was shown to be important in
cytoskeletal organization.
In particular, for the Notch - Delta/Jagged signaling pathway, the genes for
ligand of Notch receptor Jagged1, as well as the basic helix–loop–helix (bHLH)
transcriptional factor Hey1, were found to be upregulated upon adherent
meningococci infection (Table 2). Previously, expression of Jagged1 was found to be
significantly increased in injured vascular cells and Jagged/Notch activity was also
observed to be involved in regulation of cell-cell and cell-matrix interactions (Lindner,
et al., 2001). The transcriptional factor Hey1 was identified earlier as a link between
Jagged1/Notch and the TGF-β/Smad3 signaling pathways and the potential of TGF-β
to regulate and/or switch cellular differentiation programs was previously shown
(Zavadil, et al., 2004). Coordinated activation of Notch, Wnt, and TGF-β signaling
10
pathways was found also in regulation of osteoblast differentiation/maturation
(Zamurovic, et al., 2004).
Furthermore, within the Wnt/β-catenin signaling pathway we found two upregulated proteins: the sex determining region Y-box 17 (Sox17) and dickkopf 1
(Dkk1), as shown in Table 2. Sox17 is a component of Wnt signaling pathway that
was reported to physically interact with β-catenin and to potentiate its transcriptional
activation of target genes (Sinner, et al., 2004). Secreted Dickkopf protein acts as a
potent inhibitor of Wnt signaling (Mao, et al., 2001) but simultaneously high level of
this protein allows the cells to re-enter the cell cycle (Gregory, et al., 2003).
Within the TGF-β/Smad signaling pathway many up-regulated genes were
found: an inhibitor Smad7, inhibitors of differentiation (Id1, Id2, Id3) and
transmembrane glycoprotein BAMBI. TGF-β is an important member of a cytokine
superfamily that control cell fates, including cell cycle arrest, differentiation, and
apoptosis. The expression of the inhibitor of TGF-β pathway - Smad7- gene is
induced by TGF-β itself (Nakao, et al., 1997) and by fluid shear stress acting on
endothelial cells (Topper, et al., 1997). Moreover, data from colorectal cancers
support the concept that Smad7 may be beneficial for tumorgenesis (Roberts, 2002).
Forced expression of inhibitors of differentiation (Id) induces proliferative activity in
HUVECs and overexpression of Id enhances expression of ICAM-1 and E-selectin
and induces angiogenic processes (ten Dijke, et al., 2003). Here, Id1, Id2 and Id3
proteins were found to be up-regulated in HUVEC cells infected with piliated
meningococci but were down-regulated in HUVEC cells infected with non-piliated
meningococci. Further gene found upregulated here (Table 2), encodes the
glycoprotein BAMBI. BAMBI is activated by Wnt/β-catenin signal transduction
pathways implicated in regulating cell fate and cell proliferation. BAMBI expression is
aberrantly elevated in most colorectal carcinomas (Sekiya, et al., 2004).
The specific roles of TGF-β/Smad, Wnt/β-catenin and Notch - Delta/Jagged
signaling pathways in regulating host response in infectious diseases and especially
their cooperation are poorly understood and the results presented here suggest that
these signaling pathways may play an important role in the crosstalk between
meningococci and host cells.
Adhesion of meningococci was further found to alter expression of genes for
several other transcription factors (Table 2), such as GATA6, FoxC2, Fli-1, MAFB,
CBFβ, which belong to various protein families, together with some other up11
regulated proteins, such as the RGC-32 having a role in cell cycle activation (Badea,
et al., 1998) or ENC-1 important in cytoskeletal organization (Hernandez, et al.,
1998). The roles of these genes are often found in cell differentiation, embryogenesis
and vascular remodeling (Sokabe, et al., 2004) or associated with
inflammatory/stress-like responses (Li, et al., 2002). Moreover, adherent
meningococci promoted here down-regulation of MTSG1, which was identified as
potential tumor suppressor (Seibold, et al., 2003).
Significant differences in levels of gene expression between cells infected by
adherent and non-adherent meningoccci were observed also for a whole group of
pro-apoptotic genes. These were found to be specifically up-regulated by nonadhering meningococci and comprised genes for DNA-damage-inducible transcript 4
(DDIT4), zinc finger protein 36, C3H type-like 2 (ZFP36L2), BCL2/adenovirus E1B
19kDa interacting protein 3 (BNIP3) and caspase 7, apoptosis-related cysteine
protease (CASP7).
Further striking was the upregulation of transcription level of the gene for
adrenomedullin (ADM) only by the non-adherent bacteria. ADM is a multifunctional
peptide that has protective effects against vascular injury and apoptosis, regulates
angiogenesis and was postulated to have an important protective role and
antimicrobial activity against both pathogenic and commensal strains of Grampositive and Gram-negative bacteria. Reduced expression of this antimicrobial
peptide was, in fact, previously reported to result in reduced control of infections by
pathogenic and commensal organisms (Allaker & Kapas, 2003, Nagaya, et al., 2005).
In line with this, upregulation of ADM expression was prevented by adhesion of
meningococci to HUVEC cells.
Concluding remarks.
Large group of adherence-upregulated genes (ICAM-1, cyclooxygenase 2, Smad6,
Smad7, TGFβ1, GADD45, Jagged1 or the transcription factors Id1, Id2, Id3 and
HEY2) corresponded to endothelial flow-responsive genes which were identified
before. As was shown recently for Neisseria gonorrhoeae, pilus retraction acts as a
mechanical stimulus by activating mechanical stress–signaling pathways that alter
epithelial cell gene expression and generate a cytoprotective environment within the
12
host cell (Howie, et al., 2005). These results are in line with a previous finding that
biomechanical forces can modulate endothelial phenotype through changes in gene
expression contributing to the atheroprotective effects (McCormick, et al., 2001,
Wasserman, et al., 2002).
Alltogether, the results presented here indicate that pilus-mediated adhesion and
growth of meningococci in microcolonies on host cell surface results in signaling from
the bacteria that manipulate the host cells and probably increase the ability of the
cells to withstand apoptotic signals induced by infection and activate cytoprotective
signaling to maintain their normal functions. The mechanisms of crosstalk between
adherent N. meningitidis bacteria and host cells appear to play an important role in
the establishment of a commensal relationship of meningococci with the host in the
course of asymptomatic colonization of the nasopharynx.
In turn, detailed investigation of the pro-inflammatory and pro-apoptotic signaling
caused by freely proliferating, non-adherent meningococci, is than likely to shed more
light on the mechanisms of human vascular cell damage that appears to play a key
role in the pathogenesis of the invasive meningoccoal disease.
13
Acknowledgments
This work was supported by a collaborative research agreement between the
national Science Foundation of the Czech Republic and the INSERM (Institut
National de la santé et de la recherche médicale). The laboratory of Peter Sebo is
founded by National Science Foundation of the Czech Republic No. GACR
310/06/720, Institutional Research Concept AVOZ50200510. The laboratory of
Xavier Nassif is founded by INSERM, Université René Descartes, Paris 5.
14
References
Allaker, RP & Kapas, S (2003) Adrenomedullin and mucosal defence: interaction
between host and microorganism. Regul Pept 112: 147-152.
Badea, TC, Niculescu, FI, Soane, L, Shin, ML & Rus, H (1998) Molecular cloning and
characterization of RGC-32, a novel gene induced by complement activation in
oligodendrocytes. J Biol Chem 273: 26977-26981.
Barrett, T, Suzek, TO, Troup, DB, Wilhite, SE, Ngau, WC, Ledoux, P, Rudnev, D,
Lash, AE, Fujibuchi, W & Edgar, R (2005) NCBI GEO: mining millions of
expression profiles--database and tools. Nucleic Acids Res 33: D562-566.
Binnicker, MJ, Williams, RD & Apicella, MA (2003) Infection of human urethral
epithelium with Neisseria gonorrhoeae elicits an upregulation of host antiapoptotic factors and protects cells from staurosporine-induced apoptosis. Cell
Microbiol 5: 549-560.
Binnicker, MJ, Williams, RD & Apicella, MA (2004) Gonococcal porin IB activates NFkappaB in human urethral epithelium and increases the expression of host
antiapoptotic factors. Infect Immun 72: 6408-6417.
Carbonnelle, E, Helaine, S, Prouvensier, L, Nassif, X & Pelicic, V (2005) Type IV
pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occurring
after pilus assembly, essential for fibre stability and function. Mol Microbiol 55:
54-64.
Christodoulides, M, Makepeace, BL, Partridge, KA, Kaur, D, Fowler, MI, Weller, RO
& Heckels, JE (2002) Interaction of Neisseria meningitidis with human
meningeal cells induces the secretion of a distinct group of chemotactic,
proinflammatory, and growth-factor cytokines. Infect Immun 70: 4035-4044.
Dixon, GL, Heyderman, RS, Kotovicz, K, Jack, DL, Andersen, SR, Vogel, U, Frosch,
M & Klein, N (1999) Endothelial adhesion molecule expression and its inhibition
by recombinant bactericidal/permeability-increasing protein are influenced by
the capsulation and lipooligosaccharide structure of Neisseria meningitidis.
Infect Immun 67: 5626-5633.
Finlay, BB & Cossart, P (1997) Exploitation of mammalian host cell functions by
bacterial pathogens. Science 276: 718-725.
15
Fischer, SF, Schwarz, C, Vier, J & Hacker, G (2001) Characterization of antiapoptotic
activities of Chlamydia pneumoniae in human cells. Infect Immun 69: 71217129.
Forman, S, Linhartova, I, Osicka, R, Nassif, X, Sebo, P & Pelicic, V (2003) Neisseria
meningitidis RTX proteins are not required for virulence in infant rats. Infect
Immun 71: 2253-2257.
Gregory, CA, Singh, H, Perry, AS & Prockop, DJ (2003) The Wnt signaling inhibitor
dickkopf-1 is required for reentry into the cell cycle of human adult stem cells
from bone marrow. J Biol Chem 278: 28067-28078.
Hernandez, MC, Andres-Barquin, PJ, Holt, I & Israel, MA (1998) Cloning of human
ENC-1 and evaluation of its expression and regulation in nervous system
tumors. Exp Cell Res 242: 470-477.
Heyninck, K, Kreike, MM & Beyaert, R (2003) Structure-function analysis of the A20binding inhibitor of NF-kappa B activation, ABIN-1. FEBS Lett 536: 135-140.
Howie, HL, Glogauer, M & So, M (2005) The N. gonorrhoeae type IV pilus stimulates
mechanosensitive pathways and cytoprotection through a pilT-dependent
mechanism. PLoS Biol 3: e100.
Irizarry, RA, Hobbs, B, Collin, F, Beazer-Barclay, YD, Antonellis, KJ, Scherf, U &
Speed, TP (2003) Exploration, normalization, and summaries of high density
oligonucleotide array probe level data. Biostatistics 4: 249-264.
Kellogg, DS, Jr., Peacock, WL, Jr., Deacon, WE, Brown, L & Pirkle, DI (1963)
Neisseria Gonorrhoeae. I. Virulence Genetically Linked to Clonal Variation. J
Bacteriol 85: 1274-1279.
Lally, ET, Hill, RB, Kieba, IR & Korostoff, J (1999) The interaction between RTX
toxins and target cells. Trends Microbiol 7: 356-361.
Lee, EG, Boone, DL, Chai, S, Libby, SL, Chien, M, Lodolce, JP & Ma, A (2000)
Failure to regulate TNF-induced NF-kappaB and cell death responses in A20deficient mice. Science 289: 2350-2354.
Li, X, Massa, PE, Hanidu, A, Peet, GW, Aro, P, Savitt, A, Mische, S, Li, J & Marcu,
KB (2002) IKKalpha, IKKbeta, and NEMO/IKKgamma are each required for the
NF-kappa B-mediated inflammatory response program. J Biol Chem 277:
45129-45140.
Lindner, V, Booth, C, Prudovsky, I, Small, D, Maciag, T & Liaw, L (2001) Members of
the Jagged/Notch gene families are expressed in injured arteries and regulate
16
cell phenotype via alterations in cell matrix and cell-cell interaction. Am J Pathol
159: 875-883.
Mao, B, Wu, W, Li, Y, Hoppe, D, Stannek, P, Glinka, A & Niehrs, C (2001) LDLreceptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411: 321325.
McCormick, SM, Eskin, SG, McIntire, LV, Teng, CL, Lu, CM, Russell, CG & Chittur,
KK (2001) DNA microarray reveals changes in gene expression of shear
stressed human umbilical vein endothelial cells. Proc Natl Acad Sci U S A 98:
8955-8960.
McGuinness, BT, Clarke, IN, Lambden, PR, Barlow, AK, Poolman, JT, Jones, DM &
Heckels, JE (1991) Point mutation in meningococcal por A gene associated with
increased endemic disease. Lancet 337: 514-517.
Nagaya, N, Mori, H, Murakami, S, Kangawa, K & Kitamura, S (2005) Adrenomedullin:
angiogenesis and gene therapy. Am J Physiol Regul Integr Comp Physiol 288:
R1432-1437.
Nakagawa, I, Nakata, M, Kawabata, S & Hamada, S (2004) Transcriptome analysis
and gene expression profiles of early apoptosis-related genes in Streptococcus
pyogenes-infected epithelial cells. Cell Microbiol 6: 939-952.
Nakao, A, Afrakhte, M, Moren, A, et al. (1997) Identification of Smad7, a TGFbetainducible antagonist of TGF-beta signalling. Nature 389: 631-635.
Nakhjiri, SF, Park, Y, Yilmaz, O, Chung, WO, Watanabe, K, El-Sabaeny, A, Park, K &
Lamont, RJ (2001) Inhibition of epithelial cell apoptosis by Porphyromonas
gingivalis. FEMS Microbiol Lett 200: 145-149.
Nassif, X, Beretti, JL, Lowy, J, Stenberg, P, O'Gaora, P, Pfeifer, J, Normark, S & So,
M (1994) Roles of pilin and PilC in adhesion of Neisseria meningitidis to human
epithelial and endothelial cells. Proc Natl Acad Sci U S A 91: 3769-3773.
Nunn, DN & Lory, S (1991) Product of the Pseudomonas aeruginosa gene pilD is a
prepilin leader peptidase. Proc Natl Acad Sci U S A 88: 3281-3285.
Osicka, R, Kalmusova, J, Krizova, P & Sebo, P (2001) Neisseria meningitidis RTX
protein FrpC induces high levels of serum antibodies during invasive disease:
polymorphism of frpC alleles and purification of recombinant FrpC. Infect Immun
69: 5509-5519.
17
Pelicic, V, Morelle, S, Lampe, D & Nassif, X (2000) Mutagenesis of Neisseria
meningitidis by in vitro transposition of Himar1 mariner. J Bacteriol 182: 53915398.
Pujol, C, Eugene, E, Marceau, M & Nassif, X (1999) The meningococcal PilT protein
is required for induction of intimate attachment to epithelial cells following pilusmediated adhesion. Proc Natl Acad Sci U S A 96: 4017-4022.
Roberts, AB (2002) The ever-increasing complexity of TGF-beta signaling. Cytokine
Growth Factor Rev 13: 3-5.
Robinson, K, Taraktsoglou, M, Rowe, KS, Wooldridge, KG & Ala'Aldeen, DA (2004)
Secreted proteins from Neisseria meningitidis mediate differential human gene
expression and immune activation. Cell Microbiol 6: 927-938.
Rudel, T, Facius, D, Barten, R, Scheuerpflug, I, Nonnenmacher, E & Meyer, TF
(1995) Role of pili and the phase-variable PilC protein in natural competence for
transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A 92: 79867990.
Seibold, S, Rudroff, C, Weber, M, Galle, J, Wanner, C & Marx, M (2003) Identification
of a new tumor suppressor gene located at chromosome 8p21.3-22. Faseb J
17: 1180-1182.
Sekiya, T, Oda, T, Matsuura, K & Akiyama, T (2004) Transcriptional regulation of the
TGF-beta pseudoreceptor BAMBI by TGF-beta signaling. Biochem Biophys Res
Commun 320: 680-684.
Shirin, H, Sordillo, EM, Kolevska, TK, et al. (2000) Chronic Helicobacter pylori
infection induces an apoptosis-resistant phenotype associated with decreased
expression of p27(kip1). Infect Immun 68: 5321-5328.
Sinner, D, Rankin, S, Lee, M & Zorn, AM (2004) Sox17 and beta-catenin cooperate
to regulate the transcription of endodermal genes. Development 131: 30693080.
Sokabe, T, Yamamoto, K, Ohura, N, Nakatsuka, H, Qin, K, Obi, S, Kamiya, A &
Ando, J (2004) Differential regulation of urokinase-type plasminogen activator
expression by fluid shear stress in human coronary artery endothelial cells. Am
J Physiol Heart Circ Physiol 287: H2027-2034.
ten Dijke, P, Korchynskyi, O, Valdimarsdottir, G & Goumans, MJ (2003) Controlling
cell fate by bone morphogenetic protein receptors. Mol Cell Endocrinol 211:
105-113.
18
Thompson, SA, Wang, LL & Sparling, PF (1993) Cloning and nucleotide sequence of
frpC, a second gene from Neisseria meningitidis encoding a protein similar to
RTX cytotoxins. Mol Microbiol 9: 85-96.
Thompson, SA, Wang, LL, West, A & Sparling, PF (1993) Neisseria meningitidis
produces iron-regulated proteins related to the RTX family of exoproteins. J
Bacteriol 175: 811-818.
Topper, JN, Cai, J, Qiu, Y, et al. (1997) Vascular MADs: two novel MAD-related
genes selectively inducible by flow in human vascular endothelium. Proc Natl
Acad Sci U S A 94: 9314-9319.
Wasserman, SM, Mehraban, F, Komuves, LG, Yang, RB, Tomlinson, JE, Zhang, Y,
Spriggs, F & Topper, JN (2002) Gene expression profile of human endothelial
cells exposed to sustained fluid shear stress. Physiol Genomics 12: 13-23.
Welch, RA (2001) RTX toxin structure and function: a story of numerous anomalies
and few analogies in toxin biology. Curr Top Microbiol Immunol 257: 85-111.
Wells, DB, Tighe, PJ, Wooldridge, KG, Robinson, K & Ala' Aldeen, DA (2001)
Differential gene expression during meningeal-meningococcal interaction:
evidence for self-defense and early release of cytokines and chemokines. Infect
Immun 69: 2718-2722.
Wilson, R, Dowling, RB & Jackson, AD (1996) The biology of bacterial colonization
and invasion of the respiratory mucosa. Eur Respir J 9: 1523-1530.
Zamurovic, N, Cappellen, D, Rohner, D & Susa, M (2004) Coordinated activation of
notch, Wnt, and transforming growth factor-beta signaling pathways in bone
morphogenic protein 2-induced osteogenesis. Notch target gene Hey1 inhibits
mineralization and Runx2 transcriptional activity. J Biol Chem 279: 3770437715.
Zavadil, J, Cermak, L, Soto-Nieves, N & Bottinger, EP (2004) Integration of TGFbeta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal
transition. Embo J 23: 1155-1165.
Zhao, B, Bowden, RA, Stavchansky, SA & Bowman, PD (2001) Human endothelial
cell response to gram-negative lipopolysaccharide assessed with cDNA
microarrays. Am J Physiol Cell Physiol 281: C1587-1595.
19
Table 1
Adherence independent effect of meningococcal infection - a comparison of transcriptome of HUVEC/MC58WT - HUVECs and HUVEC/MC58ΔpilD - HUVECs.
ID Affymetrix
211506_s_at
216598_s_at
204470_at
209774_x_at
207850_at
206211_at
202638_s_at
203868_s_at
210056_at
202023_at
202150_s_at
204748_at
205289_at
202531_at
202510_s_at
208394_x_at
209099_x_at
221477_s_at
205207_at
202644_s_at
207196_s_at
201631_s_at
210512_s_at
201170_s_at
218507_at
212977_at
221009_s_at
209795_at
201473_at
204194_at
201502_s_at
209239_at
202076_at
gene
IL8
MCP1 (CCL2)
CXCL1
CXCL2
CXCL3
SELE
ICAM1
VCAM1
RND1
EFNA1
NEDD9(HEF1)
COX-2(PTGS2)
BMP2
IRF1
TNFAIP2(B94)
ESM1
JAG1
SOD2
IL6
TNFAIP3(A20)
TNIP1(ABIN1)
IER3
VEGF
BHLHB2(DEC1)
HIG2
CMKOR1(RDC1)
ANGPTL4
CD69
JUNB
BACH1
NFKBIA
NFKB1
BIRC2(IAP-2)
interleukin 8
chemokine (C-C motif) ligand 2
chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)
chemokine (C-X-C motif) ligand 2
chemokine (C-X-C motif) ligand 3
Selectin E (endothelial adhesion molecule 1)
intercellular adhesion molecule 1 (CD54), human rhinovirus receptor
vascular cell adhesion molecule 1
Rho family GTPase 1
ephrin-A1
neural precursor cell expressed, developmentally down-regulated 9
prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)
bone morphogenetic protein 2
interferon regulatory factor 1
tumor necrosis factor, alpha-induced protein 2
endothelial cell-specific molecule 1
jagged 1 (Alagille syndrome)
superoxide dismutase 2, mitochondrial
interleukin 6
tumor necrosis factor, alpha-induced protein 3
TNFAIP3 interacting protein 1
immediate early response 3
vascular endothelial growth factor
basic helix-loop-helix domain containing, class B, 2
hypoxia-inducible protein 2
chemokine orphan receptor 1
angiopoietin-like 4
CD69 antigen (p60, early T-cell activation antigen)
jun B proto-oncogene
BTB and CNC homology 1, basic leucine zipper transcription factor 1
nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105)
baculoviral IAP repeat-containing 2
4 hours
WT
up-reg.
51.90x
6.83x
45.71x
62.60x
42.85x
63.05x
16.67x
(11.17x)a
10.54x
4.00x
Pil Dup-reg.
55.18x
7.29x
69.40x
97.45x
73.45x
65.65x
27.02x
41.15x
6.59x
4.54x
(8.15x)a
(3.90x)b
2.55x
4.46x
(4.23x)a
(3.46x)a
4.59x
8.24x
7.26x
3.12x
3.51x
2.96x
22.68x
(2.85x)a
9.64x
2.54x
(2.01x)a
2.09x
(4.43x)b
3.18x
3.76x
3.72x
2.01x
(5.49x)b
5.53x
5.00x
7.33x
4.95x
4.23x
2.49x
5.73x
2.43x
6 hours
WT
up-reg.
76.96x
(10.13x)a
59.11x
(154.23x)c
(52.45x)a
117.25x
(25.62x)b
(9.33x)b
(7.30x)a
8.08x
3.55x
42.28x
9.69x
1.97x
(3.56x)a
4.41x
9.79x
1.94x
(8.83x)a
(18.11x)b
2.31x
6.80x
(3.80x)b
(8.98x)a
15.11x
(13.75x)c
(7.80x)c
2.44x
9.98x
(3.66x)b
4.06x
Pil Dup-reg.
76.72x
(10.17x)a
80.75x
(141.20x)c
(88.07x)c
99.31x
59.89x
38.03x
(4.17x)c
8.95x
2.90x
12.84x
6.32x
(4.20x)c
(8.56x)c
3.57x
4.85x
6.54x
(3.34x)b
(19.45x)a
4.28x
(6.40x )a
3.56x
12.73x
5.21x
28.32x
5.04x
(8.90x )c
(6.31x)c
2.13x
10.17x
2.78x
3.29x
20
Table 2
Specific influence of type IV pili and promotion of meningococcal microcolonies on HUVEC cell surface - a
comparison of transcriptome of HUVEC/MC58WT to HUVEC/MC58ΔpilD.
4 hours
ID
gene
Affymetrix
6 hours
WT/PilD-
WT/PilD-
up-reg.
up-reg.
transcriptional regulation and cell proliferation
209098_s_at JAG1
jagged 1 (Alagille syndrome)
2.33x
2.02x
44783_s_at
HEY1
hairy/enhancer-of-split related with YRPW motif 1
4.94x
1.73x
219743_at
HEY2
hairy/enhancer-of-split related with YRPW motif 2
3.53x
219993_at
SOX17
SRY (sex determining region Y)-box 17
2.17x
204602_at
DKK1
dickkopf homolog 1 (Xenopus laevis)
204790_at
SMAD7
3.40x
MAD, mothers against decapentaplegic homolog 7 (Drosophila)
6.17x
208937_s_at ID1
inhibitor of DNA binding 1, dominant negative helix-loop-helix protein
6.89x
4.03x
201565_s_at ID2
inhibitor of DNA binding 2, dominant negative helix-loop-helix protein
10.15x
2.07x
207826_s_at ID3
inhibitor of DNA binding 3, dominant negative helix-loop-helix protein
3.21x
(2.83x)a
203304_at
BAMBI
BMP and activin membrane-bound inhibitor homolog (Xenopus laevis)
2.10x
210002_at
GATA6
GATA binding protein 6
1.95x
214520_at
FOXC2
forkhead box C2 (MFH-1, mesenchyme forkhead 1)
2.02x
204236_at
FLI1
Friend leukemia virus integration 1
-3.56x
218559_s_at MAFB
v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian)
2.03x
218723_s_at RGC32
response gene to complement 32
2.54x
201730_s_at TPR
translocated promoter region (to activated MET oncogene)
(2.95x)d
(1.93x)d
201341_at
ectodermal-neural cortex (with BTB-like domain)
2.66x
(2.27x)b
ENC1
212096_s_at MTSG1
mitochondrial tumor suppressor gene 1
(1.94x)
(3.83x)d
204094_s_at KIAA0669
KIAA0669 gene product
1.89x
3.45x
202370_s_at CBFB
core-binding factor, beta subunit
201324_at
epithelial membrane protein 1
EMP1
d
2.33x
1.81x
2.48x
apoptosis, cell adhesion, inflammatory response, hypoxia and other groups of genes
205051_s_at KIT
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
(4.57x)d
209304_x_at GADD45B
growth arrest and DNA-damage-inducible, beta
2.25x
202887_s_at DDIT4
DNA-damage-inducible transcript 4
-4.52x
-2.96x
201368_at
ZFP36L2
zinc finger protein 36, C3H type-like 2
-2.09x
-2.47x
201849_at
BNIP3
BCL2/adenovirus E1B 19kDa interacting protein 3
-6.93x
207181_s_at CASP7
caspase 7, apoptosis-related cysteine protease
-2.22x
213844_at
homeo box A5
(2.00x)d
HOXA5
(2.11x)d
209288_s_at CDC42EP3
CDC42 effector protein (Rho GTPase binding) 3
-2.03x
-(2.23x)b
206336_at
chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2)
-4.51x
-(4.38x)a
201865_x_at NR3C1
nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)
2.51x
222162_s_at ADAMTS1
3.32x
201695_s_at NP
a disintegrin-like and metalloprotease (reprolysin type) with
thrombospondin type 1 motif, 1
nucleoside phosphorylase
216841_s_at SOD2
superoxide dismutase 2, mitochondrial
-2.09x
221841_s_at KLF4
Kruppel-like factor 4 (gut)
2.91x
218711_s_at SDPR
serum deprivation response (phosphatidylserine binding protein)
-(2.51x)a
-3.51x
201739_at
SGK
serum/glucocorticoid regulated kinase
1.89x
3.68x
202912_at
ADM
adrenomedullin
-5.14x
-8.89x
221009_s_at ANGPTL4
angiopoietin-like 4
-3.07x
-5.90x
218507_at
HIG2
hypoxia-inducible protein 2
-2.05x
-4.85x
202668_at
EFNB2
ephrin-B2
2.67x
CXCL6
207543_s_at P4HA1
204011_at
SPRY2
procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase),
alpha polypeptideI
sprouty homolog 2 (Drosophila)
(14.48x)a
2.23x
-4.58x
-2.12x
3.27x
6.67x
21
Table legends
Table 1
In the first hour no significant changes in gene expression level was found. There
was a large up-regulation in HUVEC gene expression in comparison between
HUVECs infected with MC58 WT resp. MC58ΔpilD strains to noninfected HUVECs
after 4 and 6 hour of incubation with meningococci. The p-value below 0.05 and a
ratio over 1.7 times were used as thresholds for inclusion into the list.
All data including Affymetrix .CEL files are available at NCBI Gene Expression
Omnibus (http://www.ncbi.nlm.nih.gov/geo/) GEO Series accession number
GSE4646
[ a p-val = 0,05-0,10;
b
p-val = 0,10-0,20;
c
low signal ]
Table 2
In the first hour no significant changes in gene expression level was found. There
were found alterations in HUVEC gene expression in comparison between MC58 WT
and MC58ΔpilD strains after 4 and 6 hour of incubation with Neisseria meningitidis.
Most of influenced genes were up-regulated, only a few of them were down-regulated
in comparison to gene expression of noninfected HUVEC cells. The p-value below
0.05 and a ratio over 1.7 times were used as thresholds for inclusion into the list.
All data including Affymetrix .CEL files are available at NCBI Gene Expression
Omnibus (http://www.ncbi.nlm.nih.gov/geo/) GEO Series accession number
GSE4646
[ a p-val = 0,05-0,10;
b
p-val = 0,10-0,20;
d
down-regulated gene]
22