Neisseria gonorrhoeae pilus attenuates cytokine response of human fallopian tube explants.

Abstract

A role for pilus during attachment of Neisseria gonorrhoeae to epithelia of the female reproductive tract is currently assumed. However, Pil⁻ gonococci have been observed during infection of the reproductive tract, which prompted us to examine the effect of pili on the dynamics of infection and the inflammatory responses of mucosal explants of the human fallopian tube.
Mucosal explants were infected in vitro with Opa negative Pil⁻ and Pil⁺N. gonorrhoeae strains.
Piliation enhanced gonococcal adherence to the epithelium within 3 h of infection (P < 0.05) but thereafter did not offer advantage to gonococci to colonize the epithelial cell surface (P > 0.05). No differences were found between the strains in numbers of gonococci inside epithelial cells. Pil⁻ bacteria induced higher levels (P < 0.05) of IL-1β, TNF-α, GM-CSF, MCP-1, and MIP-1β than Pil⁺ bacteria. There were no differences between both strains in LOS pattern, and Pil expression did not change after coincubation with mucosal strips.
Results show that gonococcal invasion of the human fallopian tube can occur independently of pilus or Opa expression, and suggest that pilus, by inhibition of several key elements of the initial inflammatory response, facilitates sustained infection of this organ.

Full text

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Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2012, Article ID 491298, 7 pages
doi:10.1155/2012/491298
Research Article
Neisseriagonorrhoeae PilusAttenuates CytokineResponseof
HumanFallopian TubeExplants
LuisVelasquez,1KatherineGarc´ ıa,1Francisco Morales,1John E.Heckels,2PedroOrihuela,1
PaulaI.Rodas,1MyronChristodoulides,2and Hugo Cardenas1
1Facultad de Qu´ ımica y Biolog´ ıa, Universidad de Santiago de Chile, Casilla 40 Correo 33, 9170022 Santiago, Chile
2Neisseria Research Group, Sir Henry Wellcome Laboratories, Division of Infection, Inflammation and Immunity,
University of Southampton Medical School, Southampton SO16 6YD, UK
Correspondence should be addressed to Hugo Cardenas, hugo.cardenas@usach.cl
Received 4 July 2011; Revised 10 October 2011; Accepted 16 October 2011
Academic Editor: Lori Snyder
Copyright © 2012 Luis Velasquez et al. This is an open access article distributed under the Creative Commons AttributionLicense,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. A role for pilus during attachment of Neisseria gonorrhoeae to epithelia of the female reproductive tract is currently
assumed. However, Pil−gonococci have been observed during infection of the reproductive tract, which prompted us to examine
the effect of pili on the dynamics of infection and the inflammatory responses of mucosal explants of the human Fallopian tube.
Methods.MucosalexplantswereinfectedinvitrowithOpanegativePil−andPil+N.gonorrhoeaestrains.Results.Piliationenhanced
gonococcal adherence to the epithelium within 3h of infection (P < 0.05) but thereafter did not offer advantage to gonococci to
colonize the epithelial cell surface (P > 0.05). No differences were found between the strains in numbers of gonococci inside
epithelial cells. Pil−bacteria induced higher levels (P < 0.05) of IL-1β, TNF-α, GM-CSF, MCP-1, and MIP-1β than Pil+bacteria.
There were no differences between both strains in LOS pattern, and Pil expression did not change after coincubation with mucosal
strips. Conclusions. Results show that gonococcal invasion of the human Fallopian tube can occur independently of pilus or Opa
expression, and suggest that pilus, by inhibition of several key elements of the initial inflammatory response, facilitates sustained
infection of this organ.
1.Introduction
Neisseria gonorrhoeae (NGO) is an exclusive pathogen of
humansthatinfectsthemucosalepitheliaofthemaleurethra
and the female lower genital tract. It is the etiological
agent of gonorrhea, and, for women in particular, infection
can spread into the upper reproductive tract, including the
Fallopian tubes (FTs). Gonococcal interactions with the FT
epitheliuminitiatesalpingitis,aninflammatoryresponsethat
can lead to FT damage and infertility. In the FT, evidence
from the classical papers of McGee and colleagues using
a culture model of ex vivo FT organ explants suggested
that gonococcal interactions were mediated by expression of
type IV pilus and the phase-variable colony opacity (Opa)
protein adhesins [1, 2]. Moreover, evidence gathered with
many isolated human cell lines has also shown that in-
vasion of host cells by gonococci involves initial attachment
mediated primarily by pili [3–9] which leads to intracellular
cortical plaque formation [10] with cooperation with Opa
proteins leading to bacterial internalization and/or traversal
of epithelial barriers [11–15].
In the course of research exploring the influence of con-
traceptivesongonococcalinfectionofthehumanFT[16],we
usedaclinicalisolatethatafterwardswasphenotypicallycha-
racterized as a Pil−Opa+strain [17]. Attachment and inter-
nalization of this particular strain into the oviductal epithe-
lium were observed from the first minutes of exposure and
were followed by the release of proinflammatory cytokines
such as IL-1α, IL-1β, and TNF-α [17]. It has been suggested
that in addition to mediating bacterial interactions, pili also
have a role in innate immunostimulation by influencing
the cytokine response induced during infection [18]. In the
current study, we examined the correlation between pilus
expression in gonococcal variants lacking Opa protein on
bacterial interactions with FT mucosal explants and the
induction of cytokine secretion.

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2.MaterialsandMethods
2.1. Neisseria gonorrhoeae Strains. Neisseria gonorrhoeae
(NGO) strains P9, variant 1 (Pil−Opa−), and variant 2
(Pil+Opa−) [19] were grown on GC agar at 37◦C in 5%
(v/v) CO2. The strains were derived from the original P9 iso-
late [20, 21], and the strain phenotype was confirmed by ex-
amining colony morphology with a stereomicroscope and
with SDS-PAGE analysis and immunoblotting with specific
monoclonal antipilus antibody SM1 [22] and anti-Opa
protein antibody B33 [14], both before and after coincuba-
tion with tubal mucosal explants.
2.2.MiniscaleLOSExtractionandTricine-SDS-PAGEAnalisis.
To verify LOS integrity in the two NGO strains, LOS samples
were extracted and visualized by subjecting proteinase K
digests of whole-cell lysates to Tricine SDS-PAGE [23, 24].
After electrophoresis, the gel was fixed in a solution contain-
ing 40% ethanol and 5% acetic acid overnight followed by
silver staining [25].
2.3. Human Fallopian Tube Explants. Fallopian tubes (FTs)
were obtained after informed consent from 13 fertile donors
submitted to gynecological surgery for reasons unrelated
to this study. Exclusion criteria were sexually transmitted
disease during the last year and no history of pelvic inflam-
matory disease. Protocols were approved by the Ethics Com-
mittees of the Universidad de Santiago de Chile and the
Hospital San Jose (Santiago, Chile) where the surgeries were
carried out. Organs were processed immediately after re-
moval as previously described [16]. Briefly, after muscle dis-
section, mucosal strips were cut into 1cm3segments and
culturedinDulbecco’smodifiedEagle’smedium(DMEM,Hi
Clone) for 3h before infection with NGO.
2.4. Infection of Human FT Explants with NGO. Mucosal ex-
plants were infected with 100μL of NGO suspension (3 ×
106cfu/mL) in DMEM as previously described [17]. Ad-
herent and internalized gonococci in the mucosal strips were
examined under confocal microscopy (Axiovert 100M Zeiss
laser scanning microscope) and counted as described previ-
ously [16]. Briefly, tissues for microscopy were fixed in cold
4% (v/v) paraformaldehyde in PBS, pH 7.4 (Winkler, Santi-
ago,Chile)for1hbeforetransferto10%(w/v)(1h)and30%
(w/v)(overnight)sucrose.Theyweremountedinembedding
compound (Cryo-M-Bed, Bright Instruments Co. Ltd.,
Huntingdon, UK) and frozen at −20◦C. Sections of 6μm
were cut using a Bright Starlet Cryostat, and gonococci were
stained with a rabbit antigonococcal antiserum and fluo-
rescein-conjugated anti-rabbit IgG.
2.5. Cytokine Analysis. Samples from the culture media were
taken at intervals up to 24h of coincubation and the secre-
tion of cytokines IL-1β, IL-6, TNF-α, IL-10, TGF-β, IL-8,
GM-CSF, MCP-1, MIP-1β, and RANTES was quantified by
sandwich immunoassays as described previously [26].
2.6. Statistical Analysis. Statistical analysis was done by
ANOVA or t-test as required, using Minitab Software
Table 1: Attachment of NGO variants to human FT explant epithe-
lium. FT explants were infected with Pil−Opa−NGO (n = 7)
and Pil+Opa−(n = 6), and 16 fields were examined for each ex-
plant at each time point. In each field, gonococci were counted
within a continuous epithelial strip of at least 150 cells. The data
are presented as the mean and SEM of the number of gonococci
observed, attached, or internalized.∗denotes a significantly (P <
0.05) higher number of attached Pil+Opa−NGO compared to the
Pil−Opa−variant at 3h.
Time (h)
Attached
Pil−Opa−
2.6 ± 0.6
4.1 ± 1.1
4.2 ± 1.1
6.8 ± 0.9
Pil+Opa−
7.6 ± 2.6∗
6.3 ± 1.4
5.8 ± 1.3
6.8 ± 2.0
3
6
12
24
Internalised
Pil−Opa−
12.4 ± 1.6
14.4 ± 1.9
14.1 ± 1.8
25.1 ± 1.9
Pil+Opa−
18.0 ± 3.4
16.8 ± 2.4
15.0 ± 2.4
18.3 ± 3.9
3
6
12
24
(Minitab 15.1.30.0). Comparison of attached and internal-
ized gonococci between both strains was done by t-test at
each incubation time using log-transformed data to correct
for variance heterogeneity.
3.Results
Human FT explants were infected with Pil−Opa−and
Pil+Opa−NGO variants, and the numbers of adherent and
internalized bacteria were quantified up to 24h. NGO was
found attached to the epithelium and internalized at all time
points examined (Table 1), with Pil+bacteria showing a sig-
nificantly higher rate of initial adherence by 3h than the Pil−
variant (P < 0.05). Thereafter, however, no statistically signi-
ficant differences were observed between the numbers of
attached Pil+and Pil−NGO (P > 0.05). NGO invasion of
the FT epithelium was rapid, but there were no statistically
significant differences (P > 0.05) in the numbers of Pil+
and Pil−bacteria counted within the epithelium at all time
pointsexamined(Table 1).AdherenceandinvasionoftheFT
epithelium were confirmed by confocal microscopy.
Compared to uninfected FT explants, gonococcal infec-
tion resulted in the significant (P < 0.05) secretion of
key proinflammatory (IL-1β, IL-6, and TNFα), chemokine
(MCP-1, MIP-1β), and growth-factor related (MCP-1, MIP-
1β and GM-CSF) cytokines. By contrast, the secretion of IL-
8, RANTES, TGF-β, MIP-1α, or IL-10 was not significantly
increased by gonococcal infection compared to uninfected
explants (data not shown).
For those cytokines whose secretion was significantly in-
creased by gonococcal infection compared to uninfected FT
explants, secretion was influenced by pilus expression. In
general, infection with Pil−NGO resulted in significantly
higher secretion levels compared with Pil+NGO. The levels
of IL-1β secreted to the culture medium increased with both

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Journal of Biomedicine and Biotechnology3

3
6
12 24
0
0
40
120
80
0.2
0.6
0.4
0
0.8
10
30
20
40
GMCSF
∗
0
10
30
20
0
1
2
0
0.4
0.8
1.2
Time (h)
Cytokine secretion (ng/mL)Cytokine secretion (ng/mL)
Cytokine secretion (ng/mL)
Cytokine secretion (ng/mL)
Cytokine secretion (ng/mL)
Cytokine secretion (ng/mL)
Pil−
Pil−
Pil+
Pil+
TNF-α
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
MCP-1
MIP-1β
IL-6
IL-1β
3
6
12 24
Time (h)
Figure 1: Expression of pilus attenuates cytokine production by human FT explant epithelium. FT explants were infected with Pil−Opa−
NGO (n = 7) and Pil+Opa−(n = 6) and cytokine secretion measured over time. The columns represent mean cytokine levels and the
standard error for the sample group.∗denotes statistically significant (P < 0.05) increase in cytokine secretion induced by Pil−bacteria
compared to Pil+bacteria at given time point.
strains of NGO over time, but by 24h they were significantly
higher in explants infected with Pil−bacteria compared with
Pil+bacteria (P < 0.05; Figure 1). Secretion of TNF-α also
increased over time with both NGO strains, with the levels
induced by Pil−bacteria by 12hr significantly higher than
those observed with Pil+bacteria (P = 0.006), although
this difference disappeared by 24h (Figure 1). IL-6 secretion
induced by Pil−NGO was significantly higher at the earlier
time points (3 and 6h) compared with Pil+NGO (P < 0.05),
but these differences were not significant by 12h onwards
(P > 0.05).ForMCP-1,MIP-1β,andGM-CSF,infectionwith
Pil−NGO induced significantly higher amounts of secretion
when compared with Pil+NGO at all time points examined
(P < 0.05; Figure 1).
It is known that gonococcal phase and antigenic vari-
ation occur during in vitro growth [27] as well as during
experimental infection [28], so phenotypic stability of the
Pil−Opa−andPil+Opa−strainsusedinthecurrentstudywas
examined during infection of FT explants. Western blotting
for pilus and Opa protein expression of NGO recovered
fromtheFTexplantsdemonstratednochangesinphenotype
duringinfection(Figure 2).TherewerenodifferencesinLOS

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4Journal of Biomedicine and Biotechnology
0
0
12 12
250
150
100
75
50
37
25
20
15
10
(kD)
Time (hr)
Co-incubation
Pil−
Pil+
(a)
0
0
1212
250
150
100
75
50
37
25
20
15
10
(kD)
Time (hr)
Co-incubation
Opa−
Opa+
(b)
Figure2:WesternblotforPilandOpaproteinsinPil+Opa+andPil−Opa−gonococcalstrainsincubatedfor12hwithFallopiantubemucosal
strips. (a): Western blot for Pil protein. (b): Western blot for Opa protein. The two dark bands corresponding to Pil (a) and Opa (b) appear
in lanes from the Pil+Opa+strain only. Densitometry analysis showed no change in Pil and Opa expression after the co-incubation.
Pil−
Pil+
LOS
20
25
15
10
3.5
(kDa)
Figure 3: Lipooligosaccharide profiles of NGO strains used in this
study. Whole-cell lysates of gonococcal strains were treated with
proteinase K overnight and then boiled prior to Tricine SDS-PAGE
(16%). Protein molecular weight marker: Novex Sharp Standard
(Invitrogen).
expression between both NGO strains (Figure 3). Therefore
the patterns of adhesion/internalization and cytokine pro-
duction in these experiments can safely be correlated with
the presence or absence of pili.
4.Discussion
In the current study, we report an experimental in vitro ex-
amination of the role of gonococcal pili during epithelial
invasion and cytokine production at the initial stages of
infection of the human Fallopian tube. Experimental infec-
tion based on cell lines and cultures has provided a theo-
retical model for gonococcal infection in which pili have
a prominent role in the initial attachment to the host cell
[18, 29]. We observed in our study that the presence of
pili enhanced gonococcal adherence to the tubal epithelium
within 3h of infection compared with the corresponding
nonpiliated variant but thereafter did not appear to offer a
significant advantage to gonococci to colonize the epithelial
cell surface. We have shown previously that a clinical
Pil−Opa+gonococcalstrainattachedandinvadedtheepithe-
lium of tubal explants [17] and the appearance of Pil−bac-
teria during experimental infection has also been reported
[28]. In our study, pilus expression also did not confer any
advantagetothegonococcusforinvasion.Moreover,because
the strains we used in our experiments were both Opa

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Journal of Biomedicine and Biotechnology5
negative,ourdataalsosuggestedanOpa-independentmech-
anism for invasion of the tubal epithelium. The redundancy
of Opa is consistent with reports demonstrating that Opa−
variantswereasinfectiveasOpa+gonococciinhumanexper-
imental infections [30], and adhesion and phagocytosis of
Pil−Opa−gonococcal strains by human dendritic cells were
influenced by differences in their lipooligosaccharides [31].
Taken together with the reported literature, our findings
therefore suggest that mechanisms other than pilus or Opa-
mediated binding to surface molecules of the epithelial cells
operate with high efficiency during gonococci invasion of
the Fallopian tube. The exact nature of these mechanisms is
unclear, although it has been reported, using recombinant E.
coliexpressingspecificgonococcalcomponents,thatinvasion
of FT epithelia depended on the expression of combinations
of Opa, porin, and lipooligosaccharide [32]. However, it is
also likely that the endocytic properties of the oviductal epi-
thelium [33] probably contribute to uptake of gonococci.
In the current study, expression of pili appeared to
have a role in the regulation of cytokine responses follow-
ing gonococcal infection. Although both Opa−gonococcal
strains induced secretion of IL-1β, TNF-α, GM-CSF, MCP-1,
and MIP-1β, the stimulatory effects were significantly high-
er with the Pil−bacteria compared to Pil+bacteria. Thus, ex-
pression of gonococcal pili appeared to attenuate cytokine-
chemokine responses in the mucosal epithelial cells of the
human Fallopian tube, although the contribution of other
uncharacterized genetic differences cannot be categorically
ruled out. Or, alternatively, the absence of the large numbers
of surface-located pili structures results in remodeling of
the OM of the gonococcus to allow increased interactions
with the epithelial cell LOS receptors, leading to increased
cytokine production. These findings are in contrast to
other studies that have investigated the involvement of pilus
expression in determining the magnitude of the cytokine
response. For example, during infection of a human mono-
cytic cell line with Salmonella typhi, Pil+bacteria induced
higher levels of IL-6 production and NF-κB activation than
nonpiliated bacteria [34]. However, we should stress that
the cytokine pattern observed in our experiments might
not necessarily represent the in situ infection, because in
vitro mucosal explants are devoid of blood and/or tissue-as-
sociated immune effector cells [35], whose contribution to
the local cytokine pool is to be expected during the ad-
vanced stages of salpingitis when bacteria reach deep into the
subepithelial tissue.
By contrast to pilus expression, the presence of Opa pro-
tein was not a contributory factor to cytokine attenuation in
theFTepitheliumitself.However,ithasbeenshownwithhu-
man dendritic cells that Opa proteins can modulate innate
and adaptive immune responses, by directly suppressing T-
cell receptor signaling through engagement of coinhibitory
CD66a/CEACAM1receptor,therebyraisingthethresholdfor
CD4+T cell activation and proliferation to activating stimuli
[36, 37] and interfering with antibody production by pro-
moting human B-cell death [38]. Thus, Opa expression may
play an indirect role in inhibiting innate recognition during
FT infection by targeting the function of patrolling submu-
cosal dendritic cells.
In summary, our data suggest a modulatory role for gon-
ococcal pili during infection of the human tubal mucosa.
Expression of pilus enhances the initial rapid attachment of
the gonococcus to the Fallopian tube mucosal epithelium
but significantly attenuates the local production of several
inflammatory mediators. Although phase variation of pilus
and opa expression in the upper reproductive tract is likely
to be a dynamic process during interactions with the host,
it is likely that the Pil−phenotype is probably present at a
lowerpercentageofthegonococcalpopulation.Switchingoff
pilusexpressionwouldnotbebeneficialtothegonococcusin
that an excess inflammatory response contributory to LOS-
mediated stimulation could result. Thus, a consequence of
limiting the innate response of the epithelium is to reduce
immune effector cell recruitment to the site of infection.
This would add to the mechanisms that could allow repeated
gonococcal infections in the same individual without ever
generating protective immunity.
Acknowledgments
The study was supported by DICYT of USACH, FONDE-
CYT 1090589, and Proyecto BASAL FB 0807. The authors
acknowledge the skillful technical support of Mrs. Nieves
Aguirre.
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