Resistance of Neisseria Gonorrhoeae to Neutrophils

Abstract

Infection with the human-specific bacterial pathogen Neisseria gonorrhoeae triggers a potent, local inflammatory response driven by polymorphonuclear leukocytes (neutrophils or PMNs). PMNs are terminally differentiated phagocytic cells that are a vital component of the host innate immune response and are the first responders to bacterial and fungal infections. PMNs possess a diverse arsenal of components to combat microorganisms, including the production of reactive oxygen species and release of degradative enzymes and antimicrobial peptides. Despite numerous PMNs at the site of gonococcal infection, N. gonorrhoeae can be cultured from the PMN-rich exudates of individuals with acute gonorrhea, indicating that some bacteria resist killing by neutrophils. The contribution of PMNs to gonorrheal pathogenesis has been modeled in vivo by human male urethral challenge and murine female genital inoculation and in vitro using isolated primary PMNs or PMN-derived cell lines. These systems reveal that some gonococci survive and replicate within PMNs and suggest that gonococci defend themselves against PMNs in two ways: they express virulence factors that defend against PMNs’ oxidative and non-oxidative antimicrobial components, and they modulate the ability of PMNs to phagocytose gonococci and to release antimicrobial components. In this review, we will highlight the varied and complementary approaches used by N. gonorrhoeae to resist clearance by human PMNs, with an emphasis on gonococcal gene products that modulate bacterial-PMN interactions. Understanding how some gonococci survive exposure to PMNs will help guide future initiatives for combating gonorrheal disease.

Full text

Regardless of the anatomic site that is infected, Gc promotes an
inflammatory response that is characterized by the recruitment of
PMNs (Figure 1). In men, PMNs appear in urethral swabs and urine
several days after infection and immediately prior to the onset of
symptoms (Cohen and Cannon, 1999). The purulent exudate pro-
duced by infected men, described in the Bible and by Galen, is the
best-known aspect of gonorrheal disease and is reflected in the trans-
lation of “gonorrhea” from Greek as “flow of seed” (Edwards and
Apicella, 2004). The cervical secretions of women with gonorrhea
also contain PMNs (Evans, 1977). Bacteria in gonorrheal secretions
are attached to and within PMNs (Ovcinnikov and Delektorskij,
1971; Farzadegan and Roth, 1975; Evans, 1977; King et al., 1978;
Apicella et al., 1996). PMNs are the primary innate immune respond-
ers to bacterial and fungal infection and are capable of phagocytos-
ing and killing a variety of microorganisms (Borregaard, 2010). Yet
in spite of the numerous PMNs at the site of gonorrheal infection,
viable Gc can be cultured from the exudates of infected individuals
(Wiesner and Thompson, 1980), and a subset of Gc remain viable
when Gc are exposed to PMNs in vitro (see below). We interpret
these results to show that the PMN-driven innate immune response
to Gc is ineffective at clearing a gonorrheal infection. The persistence
of Gc in the presence of PMNs facilitates Gc’s long-term coloniza-
tion of its obligate human hosts, creating enhanced opportunity
for dissemination and transmission of gonorrhea. In this review we
will highlight our current knowledge about Gc resistance to PMN
clearance, a critical aspect of the virulence of Gc.
GONORRHEAL DISEASE
Gonorrhea is a major global health problem, with greater than 62
million cases estimated to occur worldwide per year (Anonymous,
2001). Numbers of reported cases in the United States have remained
at approximately 330,000 annually, but it is estimated that the actual
number is at least twice as high, and rates of gonorrhea are rapidly
increasing among men who have sex with men and young adults
(Workowski and Berman, 2010). The cause of gonorrhea is the Gram-
negative diplococcus Neisseria gonorrhoeae (the gonococcus or Gc).
Gc is a human-specific pathogen that is transmitted via close sexual
contact with an infected individual. Gonorrhea presents as an acute
urethritis in men and cervicitis in women, but the pharynx and rec-
tum can also be infected (Wiesner and Thompson, 1980). Because
of the frequently asymptomatic nature of female infection, gonor-
rhea is a major cause of pelvic inflammatory disease, characterized
by abdominal pain and tubal scarring that results in ectopic preg-
nancy and infertility; untreated infections in men also lead to sterility.
Disseminated Gc infections can cause arthritis–dermatitis syndrome,
endocarditis, and meningitis. Gc can also be vertically transmitted
during childbirth and is still a leading cause of infectious neonatal
blindness in the developing world (Wiesner and Thompson, 1980).
Gc remains a major public health problem due to rapid acquisition
of resistance to multiple antibiotics (Tapsall, 2009) and its ability
to phase and antigenically vary its surface structures, preventing
infected individuals from developing a protective immune response
and hindering development of a protective vaccine (Virji, 2009).
Resistance of Neisseria gonorrhoeae to neutrophils
M. Brittany Johnson and Alison K. Criss*
Department of Microbiology, University of Virginia, Charlottesville, VA, USA
Infection with the human-specific bacterial pathogen Neisseria gonorrhoeae triggers a potent,
local inflammatory response driven by polymorphonuclear leukocytes (neutrophils or PMNs).
PMNs are terminally differentiated phagocytic cells that are a vital component of the host
innate immune response and are the first responders to bacterial and fungal infections. PMNs
possess a diverse arsenal of components to combat microorganisms, including the production
of reactive oxygen species and release of degradative enzymes and antimicrobial peptides.
Despite numerous PMNs at the site of gonococcal infection, N. gonorrhoeae can be cultured
from the PMN-rich exudates of individuals with acute gonorrhea, indicating that some bacteria
resist killing by neutrophils. The contribution of PMNs to gonorrheal pathogenesis has been
modeled in vivo by human male urethral challenge and murine female genital inoculation
and in vitro using isolated primary PMNs or PMN-derived cell lines. These systems reveal
that some gonococci survive and replicate within PMNs and suggest that gonococci defend
themselves against PMNs in two ways: they express virulence factors that defend against
PMNs’ oxidative and non-oxidative antimicrobial components, and they modulate the ability
of PMNs to phagocytose gonococci and to release antimicrobial components. In this review,
we will highlight the varied and complementary approaches used by N. gonorrhoeae to resist
clearance by human PMNs, with an emphasis on gonococcal gene products that modulate
bacterial-PMN interactions. Understanding how some gonococci survive exposure to PMNs
will help guide future initiatives for combating gonorrheal disease.
Keywords: Neisseria gonorrhoeae, virulence factors, neutrophils, polymorphonuclear leukocytes, phagocytosis, reactive
oxygen species, antimicrobial peptides, neutrophil proteases
Edited by:
Cynthia N. Cornelissen, Virginia
Commonwealth University School of
Medicine, USA
Reviewed by:
William Shafer, Emory University
School of Medicine, USA
Rick Rest, Drexel University College of
Medicine, USA
*Correspondence:
Alison K. Criss, Department of
Microbiology, University of Virginia
Health Sciences Center, Box 800734,
Charlottesville, VA 22908-0734, USA.
e-mail: akc2r@virginia.edu
www.frontiersin.org April 2011 | Volume 2 | Article 77 | 1
Review ARticle
published: 13 April 2011
doi: 10.338 9/fmicb. 2011.00077

PMN ANTIMICROBIAL ACTIVITIES
PMNs are the most abundant white cells in the peripheral blood
of humans. They are professional phagocytes and the first line
of defense of the innate immune system (Borregaard, 2010). In
response to peripheral infection or damage, PMNs follow chem-
otactic cues to extravasate from the bloodstream and migrate
through tissues to reach the target site. Mucosal epithelial cells and
resident immune release chemokines for PMNs, including inter-
leukin-8, interleukin-6, tumor necrosis factor-α, and interleukin-1
(Borregaard, 2010). These chemokines are released during human
Gc infection (Ramsey et al., 1995; Hedges et al., 1998).
PMNs possess receptors to bind and phagocytose complement-
and antibody-opsonized particles [e.g., complement receptor 3 (CR3),
FcRs]. They can also engulf unopsonized particles through lectin-
like interactions or using receptors that are specific for ligands on
the particle surface (Groves et al., 2008). Interaction between PMNs
and a target particle results in the mobilization of different subsets
of cytoplasmic granules to the plasma or phagosomal membrane
(Figure 2). Granule fusion enables the degradation and killing of
microorganisms both intracellularly and extracellularly (Borregaard
et al., 2007). PMN mechanisms of microbial killing include pro-
duction of reactive oxygen species (ROS) via the NADPH oxidase
enzyme (the “oxidative burst”) as well as the oxygen-independent
activities of degradative enzymes and antimicrobial peptides (Table
1). Human PMN granules are classified as azurophilic or primary
granules, which contain myeloperoxidase, α-defensin peptides, and
cathepsin G, among other antimicrobial components; specific or
secondary granules containing the flavocytochrome b558 subunit of
NADPH oxidase, LL-37 cathelicidin, lactoferrin, and CR3; and gela-
tinase or tertiary granules containing gelatinase (Borregaard et al.,
2007). PMN granules release their contents in a set order. Initially,
gelatinase granule contents degrade extracellular matrix, allowing
PMNs to migrate across the tissues underlying the site of infection.
Next, the release of specific granules at the target destination increases
phagocytic potential due to presentation of CR3 on the PMN surface.
Finally, the release of both specific and azurophilic granules creates
an environment that is generally hostile to microbial survival (Lacy
and Eitzen, 2008). PMNs also release neutrophil extracellular traps
(NETs) composed of DNA, histones, and selected granule compo-
nents, which trap and kill microbes without requiring phagocytosis
(Papayannopoulos and Zychlinsky, 2009). Thus PMNs combine
oxygen-dependent and -independent mechanisms to combat intra-
cellular and extracellular microorganisms.
The fact that gonorrheal exudates contain viable Gc indicates
that PMNs are ineffective at completely clearing Gc infection. There
are two mechanisms that could explain how Gc survives PMN
challenge: Gc prevents PMNs from performing their normal anti-
microbial functions (phagocytosis, granule content release), or Gc
expresses defenses against oxidative and non-oxidative components
produced by PMNs (Figure 2). As we will discuss, there is now
substantial evidence for both mechanisms, which ultimately enable
Gc to survive within a host and be transmitted to new individuals.
MODEL SYSTEMS FOR EXAMINING PMNS DURING Gc
PATHOGENESIS
Four experimental approaches have been used to investigate the
involvement of PMNs in gonorrheal disease. Each has contributed
to our understanding of how PMNs are recruited during acute
gonorrhea and how Gc withstands this onslaught.
THE MALE URETHRAL CHALLENGE MODEL
Experimental human infection is limited to male urethral inocu-
lation, due to the potential for severe complications such as pel-
vic inflammatory disease in women with gonorrhea (Cohen and
FIGURE 1 | Gonorrhoeal exudates contain numerous PMNs with
associated Gc. Gram stain of the urethral exudate from a male with
uncomplicated gonorrhea. Some PMNs associate with single diplococci (thin
arrow), while others have multiple adherent and internalized Gc (thick arrow).
Note that the majority of PMNs in the exudate are uninfected.
A
C
B
FIGURE 2 | Cellular mechanisms of Gc survival after exposure to PMNs.
Gc (blue diplococcus) attaches to the surface of PMNs and is engulfed into a
phagosome (white oval). PMNs possess three classes of granules (1°, 2°, and
3°), each of which contains a unique subset of antimicrobial compounds.
Granules fuse with the nascent phagosome or plasma membrane to deliver
their contents to invading microorganisms. We propose two mechanisms that
allow Gc to survive after exposure to PMNs. (For illustrative purposes, only
intracellular Gc survival is depicted.) First, PMN granules release their
contents at the plasma membrane or into phagosomes containing Gc (A).
However, Gc virulence factors confer resistance to granules’ antimicrobial
compounds. Second, Gc prevents PMN granules from releasing their
contents at the plasma membrane or into phagosomes, allowing the bacteria
to avoid encountering PMN antimicrobial compounds (B). Either mechanism
would enable a fraction of Gc to survive and replicate in the presence of
PMNs (C).
Johnson and Criss Neisseria gonorrhoeae and neutrophils
Frontiers in Microbiology | Cellular and Infection Microbiology April 2011 | Volume 2 | Article 77 | 2

phagocytose and generate ROS in response to Opa-expressing Gc
akin to primary human PMNs (Bauer et al., 1999; Pantelic et al.,
2004). However, HL-60 cells do not possess the robust antimicrobial
activity associated with primary cells, due in part to the absence of
specific granules and other intracellular compartments (Le Cabec
et al., 1997).
PRIMARY PMNs
Research on the molecular mechanisms underlying Gc infection
of PMNs has mostly relied upon primary human cells, purified
from freshly isolated human blood. The abundance of PMNs in
human blood and the ease of purification make PMNs amenable to
infection with Gc in vitro. The limitations of working with primary
PMNs include their short half-life, their limited capacity for genetic
manipulation, and the person-to-person variability intrinsic to pri-
mary human cells. However, primary human PMNs have been used
to measure binding and phagocytosis of Gc, quantify Gc survival
after PMN exposure, and assess the roles of Gc virulence factors in
bacterial defense against PMNs (see below). Gc infection of murine
PMNs has also been conducted (Wu and Jerse, 2006; Soler-Garcia
and Jerse, 2007). Future studies using primary PMNs along with
cultured epithelial cells from relevant anatomic sites may provide
a means to examine the complex interactions between host cells
that occur during gonorrheal infection.
Gc interaction with PMNs is influenced by the physiological
state of the PMNs being used. Initial experimentation with primary
human PMNs utilized cells and Gc suspended in buffered saline
solutions (Densen and Mandell, 1978; Rest et al., 1982), but this
is unlikely to reflect the transmigrated, primed state of PMNs in
the genitourinary tract during acute infection. Research from the
laboratory of Dr. Richard Rest (Drexel University) demonstrated
that when PMNs were allowed to adhere to tissue culture-treated
dishes, they released granule components and bound significantly
more Gc than PMNs in suspension (Farrell and Rest, 1990). Dr.
Michael Apicella’s laboratory (University of Iowa) subsequently
developed an assay using collagen-adherent PMNs, which gener-
ated a system for studying the role of selected Gc virulence factors
in bacterial survival after PMN challenge (Seib et al., 2005; Simons
et al., 2005). We adapted the Apicella protocol to include PMN
treatment with the chemokine interleukin-8, which facilitates PMN
activation (Borregaard, 2010). We used this system to demonstrate
Gc survival inside PMNs and to identify Gc proteins that defend the
bacteria from killing by PMNs (Stohl et al., 2005; Criss et al., 2009).
Gc SURVIVAL AND REPLICATION IN THE PRESENCE OF PMNs
Although the survival of Gc in association with PMNs was once
hotly debated, there is now substantial evidence that gonococci
survive and multiply within human phagocytes. Examination of
urethral exudates by light and electron microscopy has repeatedly
shown the presence of abundant PMNs with associated and inter-
nalized Gc (Ovcinnikov and Delektorskij, 1971; Farzadegan and
Roth, 1975; King et al., 1978; Apicella et al., 1996). The fact that
viable gonococci can be cultured from urethral exudates or cervi-
cal swabs is strongly suggestive of Gc survival in the presence of
PMNs (Wiesner and Thompson, 1980). In vitro studies from the
Apicella laboratory using adherent human PMNs demonstrated
that over 50% of Gc internalized by PMNs remained viable for
Cannon, 1999). Urethral infection of male volunteers results in the
release of proinflammatory cytokines and appearance of PMNs
in the urogenital tract 2–3 days after infection, similar to what is
seen in natural cases of gonococcal urethritis (Cohen and Cannon,
1999). As in natural infections, exudates from males with experi-
mental Gc infection contain PMNs with associated Gc and occa-
sional exfoliated epithelial cells. Electron microscopic analysis of
these exudates revealed that a subset of Gc inside PMNs appear
intact, providing the initial evidence that Gc may survive within
PMN phagosomes (Ovcinnikov and Delektorskij, 1971; Farzadegan
and Roth, 1975; Apicella et al., 1996).
THE FEMALE MURINE GENITAL TRACT MODEL
Dr. Ann Jerse (Uniformed Services University of the Health
Sciences) has developed a female mouse model of Gc genital tract
infection, which allows gonorrheal infection to be examined in a
genetically tractable host. In this model, estradiol-treated mice are
inoculated vaginally with Gc, which allows over 80% of mice to
be colonized with bacteria for over 1 week. Infected mice produce
inflammatory cytokines, leading to rapid appearance of PMNs in
the genital tract (Jerse, 1999). Experimental infection of female
mice has provided insight into the selective advantage of opacity-
associated (Opa) protein expression on Gc survival and the roles of
Gc virulence factors conferring in vitro resistance to ROS and anti-
microbial peptides in in vivo infection (Jerse, 1999; Jerse et al., 2003;
Wu and Jerse, 2006; Wu et al., 2009; Cole et al., 2010). Because mice
lack the human-specific receptors and other components that are
likely to be important for gonorrheal disease, future studies could
employ mice transgenic for human proteins of interest. Inbred
mice that are transgenic for human carcinoembryonic antigen-
related cellular adhesion molecules (CEACAMs) and CD46, recep-
tors that are implicated in gonorrheal pathogenesis (Merz and So,
2000), have already been developed (Johansson et al., 2003; Gu
et al., 2010), with additional mouse strains likely to be produced
in the coming years.
IMMORTALIZED PMN-LIKE CELL LINES
The use of immortalized promyelocytic human cell lines to study
the molecular mechanisms of Gc pathogenesis provides a system
which is clonal, easy to maintain, and amenable to expression of
transgenes. As one example, the leukemic HL-60 cell line can be dif-
ferentiated into a PMN-like phenotype with retinoic acid (Collins
et al., 1977; Newburger et al., 1979). Differentiated HL-60 cells can
Table 1 | Antimicrobial components housed in PMN granules.
Granule class Granule components
Primary/azurophilic Cathepsin G, BPI, lysozyme, elastase,
myeloperoxidase (MPO), α-defensins
Secondary/specific Flavocytochrome b558, LL-37 (hCAP18), lysozyme,
gelatinase, lactoferrin, CD11b/CD18 (CR3)
Tertiary/gelatinase Flavocytochrome b558, lysozyme, gelatinase,
CD11b/CD18 (CR3)
Proteins that have been shown to have or produce antimicrobial activity against
Gc in vitro are bolded and italicized. Proteins to which Gc is resistant are
indicated in red type.
Johnson and Criss Neisseria gonorrhoeae and neutrophils
www.frontiersin.org April 2011 | Volume 2 | Article 77 | 3

The complement system is a key component of the innate
immune system comprised of more than 30 proteins. The com-
plement system can be activated by three routes: the classical, the
alternative, and the lectin pathway, but all three routes normally
proceed to proteolytic activation of the major complement protein
C3 and assembly of the membrane attack complex (Ram et al.,
2010). Gc has multiple ways of resisting the bactericidal activities
of complement in normal human serum. Gc binds the comple-
ment regulatory proteins C4b-binding protein (C4BP) and factor
H (fH) on its surface via porins and sialylated LOS (Ram et al.,
1998a,b, 2001; Gulati et al., 2005). C4BP restricts the amount of C3
which can be deposited by the classical complement pathway. fH is
a cofactor for factor I-mediated cleavage of C3b to the hemolytically
inactive iC3b. In the alternative pathway fH irreversibly dissociates
factor Bb to limit C3 deposition and subsequent C5 cleavage (Ram
et al., 2010). C4BP and fH provide defense against direct comple-
ment-mediated killing but concomitantly increase iC3b deposi-
tion on the Gc surface. iC3b is a ligand for CR3 (CD11b/CD18),
which in PMNs drives actin-dependent particle engulfment into
degradative phagolysosomes and production of ROS (Groves et al.,
2008). Although it is assumed that Gc is complement-opsonized
at mucosal surfaces, how opsonization impacts Gc survival after
PMN exposure remains to be explored.
up to 6 h, as determined by viable bacterial counts and electron
microscopy (Simons et al., 2005). Our group corroborated these
findings and directly detected viable extracellular and intracellu-
lar Gc after PMN infection, using dyes that reveal the integrity of
bacterial membranes (Criss et al., 2009). We conclude from these
studies that a fraction of Gc can survive both extracellularly and
intracellularly in the presence of PMNs.
There is evidence that Gc does not only persist within PMNs,
but also uses the PMNs as a site for replication. Pioneering studies
in the 1970s showed that Gc inside exudate-derived PMNs were
sensitive to penicillin, which only kills replicating bacteria. In the
presence of antimicrobial agents such as spectinomycin or pyocin
that cannot permeate eukaryotic membranes, numbers of PMN-
associated Gc increased over time, indicative of bacterial replication
inside exudatous and in vitro-infected PMNs (Veale et al., 1976,
1979; Casey et al., 1979, 1980, 1986). Using electron microscopy
and colony counts, the Apicella laboratory observed an increase
in Gc within collagen-adherent human PMNs over a 6-h infec-
tion, results also suggestive of intracellular replication (Simons
et al., 2005). Similarly, we used bacterial viability dyes to observe
an increase in the number of viable Gc inside PMNs over time
(Criss et al., 2009). While the advantage of Gc replicating inside
terminally differentiated cells of a limited life span is questionable,
the Apicella group showed that PMNs infected with Gc delay their
spontaneous apoptosis (Simons et al., 2006). We anticipate that
advances in cellular imaging will provide additional support for
Gc replication inside PMNs and will give insight into the timing
and extent of this event.
BINDING AND PHAGOCYTOSIS OF Gc BY PMNs
Since gonorrheal secretions contain PMNs associated with viable
intracellular and extracellular bacteria, Gc must possess factors
that promote attachment and phagocytosis by PMNs. Opsonic and
non-opsonic interactions are the two basic means of phagocytosis,
both of which may be utilized by Gc (Groves et al., 2008; Figure 3).
OPSONIC UPTAKE
The two major opsonins for PMN phagocytosis are immunoglob-
ulins and complement, which bind to Fc receptors and comple-
ment receptors such as CR3, respectively (Groves et al., 2008).
Patients with gonorrhea produce opsonic IgG and IgA directed
against Gc surface-exposed components including porin, Opa
proteins, pilin, iron-regulated outer membrane proteins, and
lipooligosaccharide (LOS) (Brooks et al., 1976; McMillan et al.,
1979; Tramont et al., 1980; Rice and Kasper, 1982; Siegel et al.,
1982; Lammel et al., 1985; Schwalbe et al., 1985). Intriguingly,
serum from individuals with no prior history of gonorrhea con-
tains opsonic IgG against Gc porin and IgM against Gc LOS iso-
types containing hexosamine; the non-Gc antigens recognized by
these antibodies are not known (Sarafian et al., 1983; Griffiss et al.,
1991). Many of the Gc surface structures that promote humoral
immune responses are phase and antigenically variable and thus
evade antibody-mediated immune surveillance (Virji, 2009). Also,
Gc secretes an IgA protease that cleaves the polymeric IgA in
mucosal secretions (Blake and Swanson, 1978). Thus complement
rather than antibodies is likely to drive the opsonic phagocytosis
of Gc by PMNs.
A B C
FIGURE 3 | Opsonic and non-opsonic phagocytosis of Gc by PMNs. (A)
Antibodies that recognize Gc surface structures opsonize the bacteria and
allow for phagocytosis via Fc receptors. The efficacy of immunoglobulin-
mediated phagocytosis is questionable given the extensive phase and
antigenic variation of Gc surface structures. (B) Gc binds factor H and C4
binding protein, resulting in opsonization of Gc with C3 and other complement
components. Gc is then phagocytosed via the CR3 receptor. Gc pili and porin
can cooperatively interact with CR3, which may mediate the non-opsonic
phagocytosis of Gc by PMNs. (C) Selected Opa proteins bind to CEACAM
family receptors expressed on PMNs, leading to non-opsonic phagocytosis of
Opa+ Gc.
Johnson and Criss Neisseria gonorrhoeae and neutrophils
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Gc DEFENSES AGAINST PMN ANTIMICROBIAL ACTIVITIES
Whether they remain extracellular or are phagocytosed by PMNs,
Gc must contend with the variety of oxidative and non-oxidative
antimicrobial components produced by PMNs (Figure 2). Gc iso-
lated directly from human material or guinea pig subcutaneous
chamber fluid display increased survival in the presence of phago-
cytes compared to Gc grown in vitro (Witt et al., 1976; Veale et al.,
1977), suggesting that Gc possesses factors necessary for defending
against phagocyte killing that are lost or altered with extended in
vitro culture. These Gc factors aid Gc in resisting the toxic activities
of PMNs in two ways. First, Gc prevents PMNs from producing or
releasing antimicrobial components. Second, Gc expresses virulence
factors that defend against these components. As we will describe,
many Gc gene products have been identified that protect Gc from
purified ROS, proteases, or antimicrobial peptides, but in most cases
their roles in defense against PMNs have not yet been investigated.
DEFENSES AGAINST OXIDATIVE DAMAGE
The major species of ROS include superoxide anion, hydrogen
peroxide, and hydroxyl radical. These ROS have different reac-
tivities and half-lives, but together they induce DNA, protein, and
cell membrane damage that can lead to cell death (Fang, 2004).
There are at least four potential sources of oxidative stress for Gc
in vivo. (1) PMN NADPH oxidase transports electrons across the
phagosomal or plasma membrane to generate superoxide, which
spontaneously dismutates to hydrogen peroxide. In PMNs, the
azurophilic enzyme myeloperoxidase uses hydrogen peroxide as a
substrate to generate hypochlorous acid (bleach; Roos et al., 2003).
Phagocytes can also produce reactive nitrogen species (RNS) such
as nitric oxide and peroxynitrite, but RNS appear to be of limited
importance in human PMN antimicrobial activity (Fang, 2004).
(2) Enzymes related to phagocyte NADPH oxidase are expressed in
epithelial cells, and the survival defect of Gc antioxidant mutants
inside primary cervical cells implies that epithelial cells may also be
an important source of oxidative stress for Gc (Wu et al., 2005, 2006;
Achard et al., 2009; Potter et al., 2009). (3) Lactobacillus species
that generate hydrogen peroxide are normally found in the vaginal
flora of women (Eschenbach et al., 1989). Women with inhibi-
tory lactobacilli are less likely to be infected with Gc (Saigh et al.,
1978), and lactobacilli inhibit Gc growth in vitro (Saigh et al., 1978;
Zheng et al., 1994; St Amant et al., 2002). However, it appears that
effects of lactobacilli on Gc may be independent of hydrogen per-
oxide production, since mucosal secretions can effectively quench
lactobacilli-derived ROS (O’Hanlon et al., 2010). (4) Gc generate
ROS during aerobic respiration, although this may be less of an
issue in vivo, where the oxygen tension in the genitourinary tract
is low (Archibald and Duong, 1986). Gc defenses against oxidative
stress involve manipulation of the PMN oxidative burst, detoxifying
or repair of oxidative damage, and transcriptional upregulation of
antioxidant gene products (Figure 4A).
Gc manipulation of the PMN oxidative burst
In the absence of Opa protein expression, Gc fails to induce the PMN
oxidative burst (Rest et al., 1982; Virji and Heckels, 1986; Fischer and
Rest, 1988; Criss and Seifert, 2008). Even in the presence of Opa+
Gc that induce ROS production in PMNs, the magnitude of ROS
production is small relative to stimuli such as phorbol esters or other
NON-OPSONIC UPTAKE
In the absence of antibodies or complement, efficient binding and
engulfment of Gc by PMNs is achieved via expression of colony
Opa proteins (King and Swanson, 1978; Virji and Heckels, 1986;
Fischer and Rest, 1988). Opa proteins, formerly known as “pro-
tein II,” are a family of closely related, 20–30 kD outer membrane
proteins that facilitate Gc binding and internalization by human
cells, including PMNs (Sadarangani et al., 2011). Gc strains possess
approximately 11 opa genes encoding 7–8 antigenically distinct
Opa proteins (Connell et al., 1990; Dempsey et al., 1991). Each opa
gene is phase-variable due to slipped-strand mispairing in a pen-
tameric nucleotide repeat that places the gene in our out of frame
(Murphy et al., 1989), such that individual Gc can express zero, one,
or any possible combination of Opa proteins. Differential expres-
sion of Opa proteins can influence bacterial tropism for host cell
types and provides a mechanism of immune evasion (Sadarangani
et al., 2011).
Opacity-associated proteins bind heparan sulfate proteogly-
cans (HSPGs) and/or CEACAMs. Only those Opa proteins that
bind CEACAMs are reported to influence Gc interactions with
PMNs (Sadarangani et al., 2011). The Opa-binding CEACAMs
on PMNs are CEACAM1, CEACAM3, and CEACAM6, with
CEACAM3 expression exclusively restricted to PMNs. CEACAM1
and CEACAM3 are transmembrane proteins, while CEACAM6
possesses a glycosylphosphatidylinositol anchor (Gray-Owen and
Blumberg, 2006). Binding of Opa proteins to any of the three
CEACAMs results in Gc internalization, but via different signal-
ing events (McCaw et al., 2004).
Opacity-associated protein expression is selected for in the male
urethra, the female cervix during the follicular phase of the men-
strual cycle, and in the murine cervix (James and Swanson, 1978;
Swanson et al., 1988; Jerse et al., 1994; Jerse, 1999). However, Opa−
Gc survives better after exposure to PMNs in vitro than isogenic
Opa+ Gc (Rest et al., 1982; Virji and Heckels, 1986; Criss et al., 2009).
Opa protein expression increases Gc phagocytosis by PMNs and
stimulates PMN ROS production, and both factors may influence
bacterial survival after exposure to PMNs (Rest et al., 1982; Fischer
and Rest, 1988).
Gc surface structures other than Opa proteins may contribute to
adherence and phagocytosis by PMNs. Pili and porin cooperatively
interact with CR3 on cervical epithelial cells (Edwards et al., 2002).
It is not known if this interaction occurs on PMNs, but if it were to
occur, it would drive non-opsonic uptake of Gc by PMNs. In vitro
studies suggested that “type 1,” virulent, piliated Gc were resistant to
phagocytosis and killing by PMNs compared to “type 4,” avirulent,
non-piliated bacteria that arise after extensive laboratory passage
(Ofek et al., 1974; Dilworth et al., 1975). We now know that type
1 and type 4 Gc vary in Opa expression as well as piliation, both
of which could have contributed to these observations. Purified
porins also decrease PMN actin polymerization, which may reduce
the phagocytosis of Gc by PMNs (Bjerknes et al., 1995). Serogroup
C strains of N. meningitidis with lacto-N-neotetraose (LNnT) on
LOS are phagocytosed by neutrophils in an opsonin-independent
manner (Estabrook et al., 1998); it has not been examined whether
this LOS epitope on Gc affects phagocytosis by PMNs. Together,
the combinatorial expression of Opa proteins, pili, porin, and LOS
modulate Gc binding and internalization by PMNs.
Johnson and Criss Neisseria gonorrhoeae and neutrophils
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play a significant role in protection against oxidative stress (Tseng
et al., 2001). In comparison, KatA is crucial to Gc defense against
ROS. Gc has approximately 100-fold higher levels of catalase than E.
coli (Hassett et al., 1990). Disruption of katA significantly reduces Gc
survival to hydrogen peroxide and superoxide in vitro (Johnson et al.,
1993; Soler-Garcia and Jerse, 2004; Stohl et al., 2005) and reduces
the survival of some strains of Gc in the female murine genital tract
(Wu et al., 2009). Gc also has high peroxidase Gc activity due to the
periplasmic cytochrome c peroxidase encoded by ccp (Archibald and
Duong, 1986). ccp mutant Gc show slight sensitivity to hydrogen
peroxide, which is markedly enhanced when katA is also inactivated
(Turner et al., 2003). Gc also imports Mn(II) into its cytoplasm via the
MntABC transporter, where it scavenges superoxide and hydrogen
peroxide by a mechanism independent of SodB and catalase (Tseng
et al., 2001). This system is similar to the manganese transport system
in Lactobacillus plantarum (Archibald and Duong, 1984).
Gc can also repair oxidative damage to proteins and DNA. Gc
expresses two forms of methionine sulfoxide reductase, which reverses
the oxidation of methionine residues in proteins. The MsrA pro-
tein is localized to the cytoplasm, while MsrB is secreted to the outer
membrane. A msrAB mutant is more sensitive to hydrogen peroxide
and superoxide in vitro than its wild-type parent (Skaar et al., 2002).
bacteria (Simons et al., 2005). Gc utilizes three mechanisms to reduce
the amount of ROS produced by PMNs. First, exposure to lactate that
is released from PMNs undergoing glycolysis stimulates the rate of
Gc oxygen consumption, reducing the amount available to PMNs as
a substrate for NADPH oxidase (Britigan et al., 1988). Second, puri-
fied Gc porin inhibits PMN ROS production in response to Gc, yeast
particles, and latex beads (Lorenzen et al., 2000), but not formylated
peptides (Haines et al., 1988; Bjerknes et al., 1995). Whether porin has
this effect in the context of whole Gc bacteria remains to be examined.
Third, we reported that Opa− Gc suppresses the PMN oxidative burst
induced by serum opsonized staphylococci and formylated peptides
by a process requiring bacterial protein synthesis and bacteria-PMN
contact; the bacterial products mediating this effect are not known
at this time (Criss and Seifert, 2008).
Detoxification and repair of oxidative damage
Bacteria respond to oxidative stress by catalysis of superoxide to
hydrogen peroxide by superoxide dismutase (SOD), which is then
converted to water and molecular oxygen by catalases and peroxi-
dases (Seib et al., 2006). Gc possesses a single cytoplasmic superoxide
dismutase (SodB), one cytoplasmic catalase (KatA), and several genes
annotated as peroxidases. SodB activity is low in Gc and does not
A B
FIGURE 4 | Mechanisms of Gc survival after exposure to antimicrobial
compounds produced by PMNs. (A) Resistance to oxidative damage. PMN
NADPH oxidase generates superoxide (O2
−) and hydrogen peroxide (H2O2) from
O2, which are converted to hypochlorous acid (HOCl) by myeloperoxidase
(MPO). Gc prevents PMNs from generating ROS by lactate-mediated increase in
Gc O2 consumption and suppression of NADPH oxidase activity by porin or
as-yet unidentified factors. Gc scavenges ROS through the activities of MntABC,
superoxide dismutase (SodB), catalase (KatA), and cytochrome c peroxidase
(Ccp). Gc can also repair damage due to ROS through DNA repair enzymes
(RecN), protein reductases (MsrA/B), and other proteins (Ngo1686). (B)
Resistance to PMN non-oxidative damage. Gc pili and/or porins prevent PMN
granules from releasing non-oxidative antimicrobial components. LOS protects
Gc outer membrane proteins such as Opa and porin from proteolysis by
cathepsin G. Sialylation of LOS by Lst and PEA modification of LOS by LptA
increases bacterial resistance to cathepsin G and other antimicrobials. The MisR/
MisS two-component regulator increases expression of LptA and other gene
products that confer resistance to PMN non-oxidative damage. Ngo1686 and
RecN also protect Gc from PMN non-oxidative damage. The MtrCDE and FarAB
efflux pumps export cationic antimicrobial peptides (CAMPs) and long-chain fatty
acids (FA) from the Gc cytoplasm, respectively. In most cases, the contribution
of these virulence factors to Gc survival after exposure to PMNs remains to be
determined.
Johnson and Criss Neisseria gonorrhoeae and neutrophils
Frontiers in Microbiology | Cellular and Infection Microbiology April 2011 | Volume 2 | Article 77 | 6

permeability-increasing protein (“hCAP57”), cathepsin G protease,
and LL-37 antimicrobial peptide (Casey et al., 1985; Shafer et al.,
1986, 1998). Unlike many Gram-negative bacteria, Gc are highly
resistant (>0.2 mg/ml) to another class of antimicrobial peptides,
the defensins (Qu et al., 1996), although the observed resistance
varies depending on experimental conditions used (Porter et al.,
2005). Of PMN non-oxidative granule components, cathepsin G
and LL-37 have been the most actively studied for their effects on Gc.
Cathepsin G is a highly cationic serine protease which resides
in PMN azurophilic granules. It enzymatically cleaves Gc outer
membrane proteins including porin and Opa proteins (Rest and
Pretzer, 1981; Shafer and Morse, 1987). However, heat and protease
inhibitors do not impede cathepsin G’s ability to kill Gc in vitro,
indicating its antigonococcal activity is independent of its proteo-
lytic activity (Shafer et al., 1986). Cathepsin G can insert into Gc
membranes, but killing does not appear to be due to changes in
membrane permeability; instead, cathepsin G may impede pepti-
doglycan biosynthesis (Shafer et al., 1990).
LL-37 is the active form of an 18 kD protein precursor
(“hCAP18”) which resides in specific granules. hCAP-18 is proteo-
lytically processed to LL-37 by the azurophilic granule protein pro-
teinase-3 (Sorensen et al., 2001). hCAP-18/LL-37 is also synthesized
by mucosal epithelial cells, and is readily detected in cervicovaginal
secretions (mean LL-37 concentration of 10 μg/ml) and seminal
plasma (mean hCAP-18 concentration of 86 μg/ml; Malm et al.,
2000; Tjabringa et al., 2005). Gc infection increases the levels of
hCAP18/LL-37 by two- to four-fold in cervical and urethral washes
(Porter et al., 2005; Tjabringa et al., 2005). These concentrations of
LL-37 would be sufficient to exert antibacterial activity on Gc, since
the mean inhibitory concentration of LL-37 for Gc is 6 μg/ml (Shafer
et al., 1998). The antigonococcal mechanism of action of LL-37
remains enigmatic and may be related to its ability to form pores
that disrupt the integrity of bacterial membranes (Brogden, 2005).
Although Gc are susceptible to cathepsin G and LL-37 in vitro,
the ability of some percentage of Gc to survive PMN exposure
suggests that the bacteria have evolved mechanisms to counter
these antimicrobial components. These mechanisms involve direct
modulation of PMN granule release, changes to the Gc surface to
resist non-oxidative antimicrobial components, and active export
of these components (Figure 4B).
Modulation of PMN granule release
Both pili and porin have been reported to reduce PMN granule
fusion with the plasma membrane or phagosomes. When added
to primary PMNs, purified porins inhibit azurophilic and specific
granule exocytosis (Bjerknes et al., 1995; Lorenzen et al., 2000).
“Type 1,” piliated Gc was also reported to inhibit azurophilic gran-
ule exocytosis relative to “type 4,” non-piliated Gc, but additional
surface structures expressed on type 1 bacteria may have mediated
this result (Densen and Mandell, 1978). More detailed studies with
isogenic Gc strains are necessary to determine whether and how
Gc surface structures influence granule mobilization.
Modifications to the Gc surface
Gc LOS is thought to mask proteins which are degraded by cathe-
psin G, since truncation or loss of LOS results in increased binding
of cathepsin G and increased susceptibility to cathepsin G-mediated
Many Gc gene products involved in recombinational DNA repair,
base excision repair, and nucleotide excision repair participate in Gc
defense against ROS, such as the recombinase RecA and the DNA-
binding protein RecN (Davidsen et al., 2005; Stohl et al., 2005; Stohl
and Seifert, 2006; LeCuyer et al., 2010). The putative metallopro-
tease Ngo1686 helps protect Gc from hydrogen peroxide and the
lipid oxidant cumene hydroperoxide, but the cellular targets with
which Ngo1686 interacts are currently unknown (Stohl et al., 2005).
Both ngo1686 and recN mutants have significant survival defects after
exposure to primary human PMNs, but a recA mutant does not (Stohl
et al., 2005; Criss et al., 2009).
Transcriptional induction of antioxidant gene products
Gc pre-exposed to hydrogen peroxide survives PMN challenge
significantly better than unexposed Gc (Criss et al., 2009). This
finding implies that Gc possesses complex transcriptional cir-
cuitry that is important for defenses against ROS and/or PMNs.
The transcriptome of Gc exposed to sublethal concentrations of
hydrogen peroxide was defined and revealed the upregulation of
transcripts encoding RecN, Ngo1686, and other antioxidants after
oxidative challenge (Stohl et al., 2005). Antioxidant gene expres-
sion is regulated by selected transcriptional repressors. The OxyR
protein represses KatA expression, which is relieved following oxi-
dative stress in order to increase catalase production (Tseng et al.,
2003). PerR is responsive to Mn(II) levels and represses expression
of MntC, part of the Mn(II) transporter (Wu et al., 2006). Finally,
Ngo1427, a LexA homolog, represses expression of RecN, which is
relieved when a cysteine residue is oxidized (Schook et al., 2011).
PMNs PRIMARILY DIRECT NON-OXIDATIVE ANTIMICROBIAL
COMPONENTS AGAINST Gc
Although Gc has complex mechanisms for detecting oxidative damage
and responding to it, the importance of these processes in Gc survival
to PMNs appears to be limited. Mutants in katA, sodB, ccp, or mntABC,
alone or in combination, do not affect the percentage of Gc that can
survive PMN challenge (Seib et al., 2005; Criss et al., 2009). Moreover,
Gc survival is similar between normal PMNs and ROS-deficient PMNs
obtained from patients with chronic granulomatous disease (CGD;
Rest et al., 1982; Criss and Seifert, 2008), and PMNs maintained in
anoxic conditions, as are likely to be found in the upper reproductive
tract of females, are not impaired for antigonococcal activity (Casey
et al., 1986; Frangipane and Rest, 1992). Our group showed that Gc
survival was unaffected after exposure to PMNs treated with diphenyle-
neiodonium (DPI), an inhibitor of NADPH oxidase. DPI treatment or
CGD PMNs did not increase the percent survival of ngo1686 or recN
Gc, nor did it enhance survival of Opa+ Gc that induce ROS from
PMNs (Criss et al., 2009). From these results, we conclude that PMNs
primarily direct non-oxidative antimicrobial activities against Gc. The
functional redundancy in Gc antioxidant defenses may be sufficient
to counter PMN-derived ROS; alternatively, PMNs may not generate
enough ROS during infection to affect Gc survival.
DEFENSES AGAINST NON-OXIDATIVE DAMAGE
Seminal research from the Rest and Shafer laboratories indicated
that components found inside PMN granules display oxygen-
independent antigonococcal activity (Rest, 1979; Casey et al., 1985;
Rock and Rest, 1988). These components include the bactericidal/
Johnson and Criss Neisseria gonorrhoeae and neutrophils
www.frontiersin.org April 2011 | Volume 2 | Article 77 | 7

FarB cytoplasmic membrane transporter, and MtrE. Far expres-
sion is believed to be important for survival of isolates at the rectal
mucosal surface, which is rich in diet-derived fatty acids, and does
not contribute to Gc survival in the murine genital tract (Jerse et al.,
2003). How the Far system contributes to defense of Gc against
PMNs, which may release fatty acids (Huang et al., 2010), remains
to be explored.
DISCUSSION
Despite the prevalence of gonorrhea in the human population
and the abundance of PMNs during acute gonorrheal disease,
we are just beginning to understand the molecular mechanisms
underlying Gc interactions with and resistance to PMNs. There are
three overarching questions which remain currently in the field.
First, how does a subset of Gc survive PMN challenge? As we have
described, Gc possesses gene products which protect against oxida-
tive and non-oxidative components that are made by PMNs. Many
of these gene products are necessary for in vitro protection against
isolated antimicrobial components and some provide a selective
advantage in vivo. However, in many cases, it has not been investi-
gated whether these gene products also confer a survival advantage
in the context of PMN challenge. Second, how does Gc persist over
time inside PMNs, as is seen in PMNs isolated from gonorrheal exu-
dates? Although virulence-associated Gc surface structures such as
Opa proteins, pili, porin, and LOS have been highly investigated for
their biochemistry and impact on Gc-epithelial interactions, their
effects on Gc survival inside PMNs remain enigmatic. How comple-
ment or immunoglobulin opsonization affects Gc phagocytosis by
and survival inside PMNs also needs to be examined. Finally, how
and why does Gc stimulate PMN recruitment? That is, what is the
benefit of recruiting professional antimicrobial cells to the site of Gc
infection? Given the long history of Gc in the human population,
Gc could have evolved mechanisms for inhibiting PMN recruit-
ment; instead, Gc LOS and lipoproteins are strong initiators of the
host innate immune response (Massari et al., 2002; Pridmore et al.,
2003; Zughaier et al., 2004). The answer to this question remains
enigmatic, but may be revealed once we have a better understanding
of how Gc manipulates PMNs in vitro and in vivo.
Our current knowledge of Gc interactions with PMNs dem-
onstrates the impressive ability of Gc to survive PMN challenge.
Although we are just beginning to piece together the roles of many
Gc surface structures and gene products in Gc survival after PMN
exposure, we now have model systems in hand that will allow these
issues to be directly addressed. We are optimistic that continuing to
investigate the mechanisms used by Gc to defend against PMN anti-
microbial responses will shed light on how Gc has remained a fixture
in the human population for all of recorded history (Wain, 1947;
Morton, 1977). This research also has the potential to reveal novel
human and Gc targets that can be exploited for new therapeutics to
treat the ever-growing threat of highly antibiotic-resistant gonorrhea.
ACKNOWLEDGMENTS
We thank Dr. Jeffrey Tessier for obtaining the clinical sample for
the image in Figure 1. We thank Louise Ball, Joanna Goldberg, and
the two reviewers for helpful comments on the manuscript. This
work was supported in part by NIH R00 TW008042 (Alison K.
Criss) and T32 AI007046 (M. Brittany Johnson).
killing (Shafer, 1988). Two modifications of LOS impact bacte-
rial interactions with host cells and host defenses: phosphoeth-
anolamine (PEA) substitution on lipid A or the oligosaccharide, and
sialylation of the terminal Galβ1-4GlcNAc epitopes of the oligosac-
charide (Mandrell et al., 1990; Plested et al., 1999). PEA addition
to the heptose group on the beta chain of the core oligosaccharide
enhances Gc serum resistance but does not affect susceptibility to
antimicrobial peptides (Lewis et al., 2009). In contrast, PEA addi-
tion to lipid A by the LptA enzyme increases resistance to both
normal human serum and cationic antimicrobial peptides¸ indi-
cating that structural changes in LOS contribute to the ability of
gonococci to resist the bactericidal action of these innate immune
components (Lewis et al., 2009). In the related bacterium N. men-
ingitidis, expression of lptA is positively regulated by the misR/misS
two-component regulatory system (Newcombe et al., 2005; Tzeng
et al., 2008). The roles of MisR/MisS and LptA in Gc pathogenesis
remain to be examined. The gonococcal α-2,3-sialyltransferase Lst
transfers sialyl groups from host-derived CMP-N-acetylneuraminic
acid to the terminal galactose residue on the oligosaccharide of LOS
(Gilbert et al., 1996). Sialylation contributes to Gc resistance to
normal human serum as well as PMN-derived oxygen-independ-
ent antimicrobial factors (Shafer et al., 1986; Parsons et al., 1992).
Importantly, sialylated Gc are more resistant to PMNs in vitro, and
sialylation contributes to Gc survival in the murine female genital
tract (Kim et al., 1992; Rest and Frangipane, 1992; Gill et al., 1996;
Wu and Jerse, 2006). In addition to LOS, changes in other surface
components may contribute to Gc resistance to non-oxidative anti-
microbial factors. For instance, loss of Opa expression enhances Gc
resistance to serine proteases (Blake et al., 1981; Cole et al., 2010),
and N. meningitidis lacking pili (due to insertional mutagenesis of
the pilMNOPQ operon) are more resistant to the model antimi-
crobial peptide polymyxin B (Tzeng et al., 2005).
Gc export of antimicrobial components
The multiple transferable resistance (mtr) locus is a key deter-
minant of Gc resistance to antimicrobial agents (Shafer et al.,
1998). Mtr, a member of the resistance-nodulation-cell division
(RND) family of efflux pumps, is encoded by a three gene operon
designated mtrCDE. MtrC spans the periplasm to link the inner
membrane protein MtrD, the multidrug efflux transporter, with
outer membrane protein MtrE, the channel for export of antimi-
crobials to the extracellular environment (Hagman et al., 1995).
MtrCDE uses the proton motive force to export a variety of com-
pounds from the Gc cytoplasm, including antibiotics, detergents,
and antimicrobial peptides (Hagman et al., 1995; Veal et al., 2002).
mtrCDE is negatively regulated by the MtrR transcriptional repres-
sor (Hagman and Shafer, 1995) and positively regulated by the
MtrA transcriptional activator (Rouquette et al., 1999). Mutations
in mtrR and mtrA that modulate expression of MtrCDE affect Gc
resistance to antimicrobial peptides (Hagman et al., 1995; Hagman
and Shafer, 1995). MtrCDE expression promotes Gc survival in the
murine female genital tract (Jerse et al., 2003) and enhances resist-
ance to murine antimicrobial peptides (Warner et al., 2008), but
its role in defense of Gc against PMNs is unclear. Gc also uses the
FarAB efflux pump system to confer resistance to long-chain fatty
acids, independent of Mtr activity (Lee and Shafer, 1999). The Far
system is composed of the FarA membrane-spanning linker, the
Johnson and Criss Neisseria gonorrhoeae and neutrophils
Frontiers in Microbiology | Cellular and Infection Microbiology April 2011 | Volume 2 | Article 77 | 8

Fischer, S. H., and Rest, R. F. (1988).
Gonococci possessing only certain
P.II outer membrane proteins inter-
act with human neutrophils. Infect.
Immun. 56, 1574–1579.
Frangipane, J. V., and Rest, R. F. (1992).
Anaerobic growth of gonococci does
not alter their Opa-mediated interac-
tions with human neutrophils. Infect.
Immun. 60, 1793–1799.
Gilbert, M., Watson, D. C., Cunningham,
A. M., Jennings, M. P., Young, N.
M., and Wakarchuk, W. W. (1996).
Cloning of the lipooligosaccharide
alpha-2,3-sialyltransferase from the
bacterial pathogens Neisseria men-
ingitidis and Neisseria gonorrhoeae. J.
Biol. Chem. 271, 28271–28276.
Gill, M. J., Mcquillen, D. P., Van Putten, J. P.,
Wetzler, L. M., Bramley, J., Crooke, H.,
Parsons, N. J., Cole, J. A., and Smith, H.
(1996). Functional characterization of
a sialyltransferase-deficient mutant of
Neisseria gonorrhoeae. Infect. Immun.
64, 3374–3378.
Gray-Owen, S. D., and Blumberg, R. S.
(2006). CEACAM1: contact-depend-
ent control of immunit y. Nat. Rev.
Immunol. 6, 433–446.
Griffiss, J. M., Jarvis, G. A., O’Brien, J. P.,
Eads, M. M., and Schneider, H. (1991).
Lysis of Neisseria gonorrhoeae initiated
by binding of normal human IgM to a
hexosamine-containing lipooligosac-
charide epitope(s) is augmented by
strain-specific, properdin-binding-
dependent alternative complement
pathway activation. J. Immunol. 147,
298–305.
Groves, E., Dart, A. E., Covarelli, V., and
Caron, E. (2008). Molecular mecha-
nisms of phagocytic uptake in mam-
malian cells. Cell. Mol. Life Sci. 65,
1957–1976.
Gu, A., Zhang, Z., Zhang, N., Tsark, W.,
and Shively, J. E. (2010). Generation
of human CEACAM 1 transgenic
mice and binding of Neisseria Opa
protein to their neutrophils. PLoS
ONE 5, e10067. doi: 10.1371/journal.
pone.0010067
Gulati, S., Cox, A., Lewis, L. A., Michael, F.
S., Li, J., Boden, R., Ram, S., and Rice,
P. A. (2005). Enhanced factor H bind-
ing to sialylated gonococci is restricted
to the sialylated lacto-N-neotetraose
lipooligosaccharide species: implica-
tions for serum resistance and evidence
for a bifunctional lipooligosaccharide
sialyltransferase in gonococci. Infect.
Immun. 73, 7390–7397.
Hagman, K. E., Pan, W., Spratt, B. G.,
Balthazar, J. T., Judd, R. C., and Shafer,
W. M. (1995). Resistance of Neisseria
gonorrhoeae to antimicrobial hydro-
phobic agents is modulated by the
mtrRCDE efflux system. Microbiology
141(Pt 3), 611–622.
oxidative burst of human polymor-
phonuclear leukocytes. Cell. Microbiol.
10, 2257–2270.
Davidsen, T., Bjoras, M., Seeberg, E. C.,
and Tonjum, T. (2005). Antimutator
role of DNA glycosylase MutY in path-
ogenic Neisseria species. J. Bacteriol.
187, 2801–2809.
Dempsey, J. A., Litaker, W., Madhure,
A., Snodgrass, T. L., and Cannon, J.
G. (1991). Physical map of the chro-
mosome of Neisser ia gonorrhoeae
FA1090 with locations of genetic
markers, including opa and pil genes.
J. Bacteriol. 173, 5476–5486.
Densen, P., and Mandell, G. L. (1978).
Go nococ cal i nte rac tions wit h
polymo rphonu cle ar n eutrophils:
importance of the phagosome for
bactericidal activity. J. Clin. Invest. 62,
1161–1171.
Dilworth, J. A., Hendley, J. O., and
Mandell, G. L. (1975). Attachment and
ingestion of gonococci human neu-
trophils. Infect. Immun. 11, 512–516.
Edwards, J. L., and Apicella, M. A. (2004).
The molecular mechanisms used by
Neisseria gonorrhoeae to initiate infec-
tion differ between men and women.
Clin. Microbiol. Rev. 17, 965–981, table
of contents.
Edwards, J. L., Brown, E. J., Uk-Nham,
S., Cannon, J. G., Blake, M. S., and
Apicella, M. A. (2002). A co-operative
interaction between Neisseria gonor-
rhoeae and complement receptor 3
mediates infection of primary cervi-
cal epithelial cells. Cell. Microbiol. 4,
571–584.
Eschenbach, D. A., Davick, P. R., Williams,
B. L., Klebanoff, S. J., Young-Smith, K.,
Critchlow, C. M., and Holmes, K. K.
(1989). Prevalence of hydrogen per-
oxide-producing Lactobacillus species
in normal women and women with
bacterial vaginosis. J. Clin. Microbiol.
27, 251–256.
Estabrook, M. M., Zhou, D., and Apicella,
M. A. (1998). Nonopsonic phagocyto-
sis of group C Neisseria meningitidis by
human neutrophils. Infect. Immun. 66,
1028–1036.
Evans, B. A. (1977). Ultrastructural study
of cervical gonorrhea. J. Infect. Dis.
136, 248–255.
Fang, F. C. (2004). Antimicrobial reactive
oxygen and nitrogen species: concepts
and controversies. Nat. Rev. Microbiol.
2, 820–832.
Farrell, C. F., and Rest, R. F. (1990).
Up-regulation of human neutrophil
receptors for Neisseria gonorrhoeae
expressing PII outer membrane pro-
teins. Infect. Immun. 58, 2777–2784.
Farzadegan, H., and Roth, I. L. (1975).
Scanning electron microscopy and
freeze-etching of gonorrhoeal urethral
exudate. Br. J. Vener. Dis. 51, 83–91.
metabolism of phagocytosis. J. Clin.
Invest. 81, 318–324.
Brogden, K. A. (2005). Antimicrobial pep-
tides: pore formers or metabolic inhib-
itors in bacteria? Nat. Rev. Microbiol.
3, 238–250.
Brooks, G. F., Israel, K. S., and Petersen,
B. H. (1976). Bactericidal and opsonic
activity against Neisseria gonorrhoeae
in sera from patients with dissemi-
nated gonococcal infection. J. Infect.
Dis. 134, 450–462.
Casey, S. G., Shafer, W. M., and Spitznagel,
J. K. (1985). Anaerobiosis increases
resistance of Neisseria gonorrhoeae
to O2-independent antimicrobial
proteins from human polymorpho-
nuclear granulocytes. Infect. Immun.
47, 401–407.
Casey, S. G., Shafer, W. M., and Spitznagel,
J. K. (1986). Neisseria gonorrhoeae
survive intraleukocytic oxygen-inde-
pendent antimicrobial capacities of
anaerobic and aerobic granulocytes
in the presence of pyocin lethal for
extracellular gonococci. Infect. Immun.
52, 384–389.
Casey, S. G., Veale, D. R., and Smith, H.
(1979). Demonstration of intracel-
lular growth of gonococci in human
phagocytes using spectinomycin to
kill extracellular organisms. J. Gen.
Microbiol. 113, 395–398.
Casey, S. G., Veale, D. R., and Smith,
H. (1980). Intracellular survival of
Neisseria gonorrhoeae in human ure-
thral exudate. FEMS Microbiol. Lett.
8, 97–100.
Cohen, M. S., and Cannon, J. G. (1999).
Human experimentation with
Neisseria gonorrhoeae: progress and
goals. J. Infect. Dis. 179(Suppl. 2),
S375–S379.
Cole, J. G., Fulcher, N. B., and Jerse, A.
E. (2010). Opacity proteins increase
Neisseria gonorrhoeae fitness in the
female genital tract due to a factor
under ovarian control. Infect. Immun.
78, 1629–1641.
Collins, S. J., Gallo, R. C., and Gallagher,
R. E. (1977). Continuous growth and
differentiation of human myeloid
leukaemic cells in suspension culture.
Nature 270, 347–349.
Connell, T. D., Shaffer, D., and Cannon,
J. G. (1990). Characterization of the
repertoire of hypervariable regions
in the Protein II (opa) gene family of
Neisseria gonorrhoeae. Mol. Microbiol.
4, 439–449.
Criss, A. K., Katz, B. Z., and Seifert, H.
S. (2009). Resistance of Neisseria
gonorrhoeae to non-oxidative killing
by adherent human polymorphonu-
clear leucocytes. Cell. Microbiol. 11,
1074–1087.
Criss, A. K., and Seifert, H. S. (2008).
Neisseria gonorrhoeae suppresses the
REFERENCES
Achard, M. E., Hamilton, A. J., Dankowski,
T., Heras, B., Schembri, M . S.,
Edwards, J. L., Jennings, M. P., and
Mcewan, A. G. (2009). A periplasmic
thioredoxin-like protein plays a role
in defense against oxidative stress in
Neisseria gonorrhoeae. Infect. Immun.
77, 4934–4939.
Anonymous. (2001). Global Prevalence
and Inciden ce of Sel ected Curable
Sexually Transmitted Infections:
Over view and Estimates. Geneva:
World Health Organization.
Apicella, M. A., Ketterer, M., Lee, F. K.,
Zhou, D., Rice, P. A., and Blake, M.
S. (1996). The pathogenesis of gono-
coccal urethritis in men: confocal
and immunoelectron microscopic
analysis of urethral exudates from men
infected with Neisseria gonorrhoeae. J.
Infect. Dis. 173, 636–646.
Archi bald, F. S., and Duong, M. N.
(1984). Manganese acquisition by
Lactobacillus plantarum. J. Bacteriol.
158, 1–8.
Archibald, F. S., and Duong, M. N. (1986).
Superoxide dismutase and oxygen tox-
icity defenses in the genus Neisseria.
Infect. Immun. 51, 631–641.
Bauer, F. J., Rudel, T., Stein, M., and
Meyer, T. F. (1 999). Mutagenes is
of the Neisseria gonorrhoeae porin
reduces invasion in epithelial cells and
enhances phagocyte responsiveness.
Mol. Microbiol. 31, 903–913.
Bjerknes, R., Guttormsen, H. K., Solberg,
C. O., and Wetzler, L. M . (1995).
Neisserial porins inhibit human
neut rophil actin polym erization,
deg ranulati on, opsonin rece pto r
expressio n, and phagocyto sis but
prime the neutrophils to increase
their oxidative burst. Infect. Immun.
63, 160–167.
Blake, M. S., Gotschlich, E. C., and
Swanson, J. (1981). Effects of proteo-
lytic enzymes on the outer membrane
proteins of Neisseria gonorrhoeae.
Infect. Immun. 33, 212–222.
Blake, M. S., and Swanson, J. (1978).
Studies on gonococcus infection. XVI.
Purification of Neisseria gonorrhoeae
immunoglobulin A1 protease. Infect.
Immun. 22, 350–358.
Borregaard, N. (2010). Neutrophils, from
marrow to microbes. Immunity 33,
657–670.
Borregaard, N., S orensen, O. E., and
Theilgaard-Monch, K. (2007).
Neutrophil granul es: a library of
innate immunity proteins. Trends
Immunol. 28, 340–345.
Britigan, B. E., Klapper, D., Svendsen, T.,
and Cohen, M. S. (1988). Phagocyte-
derived lactate stimulates oxygen con-
sumption by Neisseria gonorrhoeae. An
unrecognized aspect of the oxygen
Johnson and Criss Neisseria gonorrhoeae and neutrophils
www.frontiersin.org April 2011 | Volume 2 | Article 77 | 9

Retinoic acid tre ated HL6 0 cel ls
express CEACAM1 (CD66a) and
phagocytose Neisseria gonorrhoeae.
FEMS Immunol. Med. Microbiol. 42,
261–266.
Papayannopoulos, V., and Zychlinsky,
A. (2009). NETs: a new strategy for
using old weapons. Trends Immunol.
30, 513–521.
Parsons, N. J., Curry, A., Fox, A. J., Jones,
D. M., Cole, J. A., and Smith, H. (1992).
The serum resistance of gonococci in
the majority of urethral exudates is
due to sialylated lipopolysaccharide
seen as a surface coat. FEMS Microbiol.
Lett. 69, 295–299.
Plested, J. S., Makepeace, K., Jennings, M.
P., Gidney, M. A., Lacelle, S., Brisson,
J., Cox, A. D., Martin, A., Bird, A.
G., Tang, C. M., Mackinnon, F. M.,
Richards, J. C., and Moxon, E. R.
(1999). Conservation and accessibil-
ity of an inner core lipopolysaccha-
ride epitope of Neisseria meningitidis.
Infect. Immun. 67, 5417–5426.
Porter, E., Yang, H., Yavagal, S., Preza,
G. C., Murillo, O., Lima, H., Greene,
S., Mahooz i, L., Klei n-Patel, M.,
Diamond, G., Gulati, S., Ganz, T., Rice,
P. A., and Quayle, A. J. (2005). Distinct
defensin profiles in Neisseria gonor-
rhoeae and Chlamydia trachomatis
urethritis reveal novel epithelial cell–
neutrophil interactions. Infect. Immun.
73, 4823–4833.
Potter, A. J., Kidd, S. P., Edwards, J.
L., Falsetta, M. L., Apicella, M. A.,
Jennings, M. P., and Mcewan, A. G.
(2009). Thioredoxin reductase is
essential for protection of Neisseria
gonorrhoeae against killing by nitric
oxide and for bacterial growth dur-
ing interaction with cervical epithelial
cells. J. Infect. Dis. 199, 227–235.
Pridmore, A. C., Jarvis, G. A., John, C. M.,
Jack, D. L., Dower, S. K., and Read, R. C.
(2003). Activation of toll-like receptor
2 (TLR2) and TLR4/MD2 by Neisseria
is independent of capsule and lipooli-
gosaccharide (LOS) sialylation but var-
ies widely among LOS from different
strains. Infect. Immun. 71, 3901–3908.
Qu, X. D., Harwig, S. S., Oren, A. M.,
Shafer, W. M., and Lehrer, R. I. (1996).
Susceptibility of Neisseria gonorrhoeae
to protegrins. Infect . Immun . 64,
1240–1245.
Ram, S., Cullinane, M., Blom, A. M.,
Gulati, S., Mcquillen, D. P., Monks, B.
G., O’Connell, C., Boden, R., Elkins, C.,
Pangburn, M. K., Dahlback, B., and Rice,
P. A. (2001). Binding of C4b-binding
protein to porin: a molecular mecha-
nism of serum resistance of Neisseria
gonorrhoeae. J. Exp. Med. 193, 281–295.
Ram, S., Lewis, L. A., and Rice, P. A. (2010).
Infections of people with comple-
ment deficiencies and patients who
modification of Neisseria gonorrhoeae
lipooligosaccharide epitope structure by
sialylation. J. Exp. Med. 171, 1649–1664.
Massari, P., Henneke, P., Ho, Y., Latz, E.,
Golenbock, D. T., and Wetzler, L. M.
(2002). Cutting edge: immune stimu-
lation by neisserial porins is toll-like
receptor 2 and MyD88 dependent. J.
Immunol. 168, 1533–1537.
McCaw, S. E., Liao, E. H., and Gray-Owen,
S. D. (2004). Engulfment of Neisseria
gonorrhoeae: revealing distinct proc-
esses of bacterial entry by individual
carcinoembryonic antigen-related cel-
lular adhesion molecule family recep-
tors. Infect. Immun. 72, 2742–2752.
McMillan, A., Mcneillage, G., and Young,
H. (1979). Antibodies to Neisseria
gonorrhoeae: a study of the urethral
exudates of 232 men. J. Infect. Dis.
140, 89–95.
Merz, A. J., and So, M. (2000). Interactions
of pathogenic neisseriae with epithe-
lial cell membranes. Annu. Rev. Cell
Dev. Biol. 16, 423–457.
Morton, R. S. (ed.). (1977). Gonorrhoea in
Earlier Times. London: W.B. Saunders.
Murphy, G. L., Connell, T. D., Barritt, D. S.,
Koomey, M., and Cannon, J. G. (1989).
Phase variation of gonococcal protein
II: regulation of gene expression by
slipped-strand mispairing of a repeti-
tive DNA sequence. Cell 56, 539–547.
Newburger, P. E., Chovaniec, M. E.,
Greenberger, J. S., and Cohen, H. J.
(1979). Functional changes in human
leukemic cell line HL-60. A model for
myeloid differentiation. J. Cell Biol.
82, 315–322.
Newcombe, J., Jeynes, J. C., Mendoza, E.,
Hinds, J., Marsden, G. L., Stabler, R. A.,
Marti, M., and Mcfadden, J. J. (2005).
Phenotypic and transcriptional char-
acterization of the meningococcal
PhoPQ system, a magnesium-sensing
two-component regulatory system
that controls genes involved in remod-
eling the meningococcal cell surface. J.
Bacteriol. 187, 4967–4975.
Ofek, I., Beachey, E. H., and Bisno, A. L.
(1974). Resistance of Neisseria gonor-
rhoeae to phagocytosis: relationship to
colonial morphology and surface pili.
J. Infect. Dis. 129, 310–316.
O’Hanlon, D. E., Lanier, B. R., Moench,
T. R. , a nd Co ne, R . A . ( 2010) .
Cervicovaginal fluid and semen block
the microbicidal activity of hydrogen
peroxide produced by vaginal lacto-
bacilli. BMC Infect. Dis. 10, 120. doi:
10.1186/1471-2334-10-120
Ovcinnikov, N. M., and Delektorskij, V. V.
(1971). Electron microscope studies of
gonococci in the urethral secretions of
patients with gonorrhoea. Br. J. Vener.
Dis. 47, 419–439.
Pantelic, M., Chen, I., Parker, J., Zhang,
P., Grunert, F., and Chen, T. (2004).
opsonophagocytosis. Infect. Immun.
60, 4439–4442.
King, G., James, J. F., and Swanson, J.
(1978). Studies on gonococcus infec-
tion. XI. Comparison of in vivo and
vitro association of Neisseria gonor-
rhoeae with human neutrophils. J.
Infect. Dis. 137, 38–43.
King, G. J., and Swanson, J. (1978).
Studies on gonococcus infection. XV.
Identification of surface proteins of
Neisseria gonorrhoeae correlated with
leukocyte association. Infect. Immun.
21, 575–584.
Lacy, P., and Eitzen, G. (2008). Control
of granule exocytosis in neutrophils.
Front. Biosci. 13, 5559–5570.
Lammel, C. J., Sweet, R. L., Rice, P.
A., Knapp, J. S., Schoolnik, G. K.,
Heilbron, D. C., and Brooks, G. F.
(1985). Antibody-antigen specificity
in the immune response to infection
with Neisseria gonorrhoeae. J. Infect.
Dis. 152, 990–1001.
Le Cabec, V., Calafat, J., and Borregaard, N.
(1997). Sorting of the specific granule
protein, NGAL, during granulocytic
maturation of HL-60 cells. Blood 89,
2113–2121.
LeCuyer, B. E., Criss, A. K., and Seifert, H.
S. (2010) Genetic characterization of
the nucleotide excision repair system
of Neisseria gonorrhoeae. J. Bacteriol.
192, 665–673.
Lee, E. H., and Shafer, W. M. (1999). The
farAB-encoded efflux pump medi-
ates resistance of gonococci to long-
chained antibacterial fatty acids. Mol.
Microbiol. 33, 839–845.
Lewis, L. A., Choudhury, B., Balthazar, J.
T., Martin, L. E., Ram, S., Rice, P. A.,
Stephens, D. S., Carlson, R., and Shafer,
W. M. (2009). Phosphoethanolamine
substitution of lipid A and resistance of
Neisseria gonorrhoeae to cationic anti-
microbial peptides and complement-
mediated killing by normal human
serum. Infect. Immun. 77, 1112–1120.
Lorenzen, D. R., Gunther, D., Pandit,
J., Rudel, T., Brandt, E., and Meyer,
T. F. (2000). Neisseria gonorrhoeae
porin modifies the oxidative burst of
human professional phagocytes. Infect.
Immun. 68, 6215–6222.
Malm, J., Sorensen, O., Persson, T., Frohm-
Nilsson, M., Johansson, B., Bjartell,
A., Lilja, H., Stahle-Backdahl, M.,
Borregaard, N., and Egesten, A. (2000).
The human cationic antimicrobial
protein (hCAP-18) is expressed in the
epithelium of human epididymis, is
present in seminal plasma at high con-
centrations, and is attached to sperma-
tozoa. Infect. Immun. 68, 4297–4302.
Mandrell, R. E., Lesse, A. J., Sugai, J. V., Shero,
M., Griffiss, J. M., Cole, J. A., Parsons, N.
J., Smith, H., Morse, S. A., and Apicella,
M. A. (1990). In vitro and in vivo
Hagman, K. E., and Shafer, W. M. (1995).
Transcriptional control of the mtr
efflux system of Neisseria gonorrhoeae.
J. Bacteriol. 177, 4162–4165.
Haines, K. A., Yeh, L., Blake, M. S., Cristello,
P., Korchak, H., and Weissmann, G.
(1988). Protein I, a translocatable ion
channel from Neisseria gonorrhoeae,
selectively inhibits exocytosis from
human neutrophils without inhibit-
ing O2-generation. J. Biol. Chem. 263,
945–951.
Hassett, D. J., Charniga, L., and Cohen, M.
S. (1990). recA and catalase in H2O2-
mediated toxicity in Neisseria gonor-
rhoeae. J. Bacteriol. 172, 7293–7296.
Hedges, S. R., Sibley, D. A., Mayo, M.
S., Hook, E. W. III, and Russell, M.
W. (1998). Cytokine and antibody
responses in women infected with
Neisseria gonorrhoeae: effects of con-
comitant infections. J. Infect. Dis. 178,
742–751.
Huang, C. B., George, B., and Ebersole,
J. L. (2010). Antimicrobial activity of
n-6, n-7 and n-9 fatty acids and their
esters for oral microorganisms. Arch.
Oral Biol. 55, 555–560.
James, J. F., and Swanson, J. (197 8).
Studies on gonococcus infection. XIII.
Occurrence of color/opacity colonial
variants in clinical cultures. Infect.
Immun. 19, 332–340.
Jerse, A. E. (1999). Experimental gonococ-
cal genital tract infection and opacity
protein expression in estradiol-treated
mice. Infect. Immun. 67, 5699–5708.
Jerse, A. E., Cohen, M. S., Drown, P. M.,
Whicker, L. G., Isbey, S. F., Seifert, H.
S., and Cannon, J. G. (1994). Multiple
gonococca l opacity prote ins are
expressed during experimental ure-
thral infection in the male. J. Exp. Med.
179, 911–920.
Jerse, A. E., Sharma, N. D., Simms, A. N.,
Crow, E. T., Snyder, L. A., and Shafer,
W. M. (2003). A gonococcal efflux
pump system enhances bacterial
survival in a female mouse model of
genital tract infection. Infect. Immun.
71, 5576–5582.
Johansson, L., Rytkonen, A., Bergman,
P., Albiger, B., Kallstrom, H., Hokfelt,
T., Agerberth, B., Cattaneo, R., and
Jonsson, A. B. (2003). CD46 in
meningococcal disease. Science 301,
373–375.
Johnson, S. R., Steiner, B. M., Cruce, D. D.,
Perkins, G. H., and Arko, R. J. (1993).
Characterization of a catalase-defi-
cient strain of Neisseria gonorrhoeae:
evidence for the significance of cata-
lase in the biology of N. gonorrhoeae.
Infect. Immun. 61, 1232–1238.
Kim, J. J., Zhou, D., Mandrell, R. E., and
Griffiss, J. M. (1992). Effect of exog-
enous sialylation of the lipooligosac-
charide of Neisseria gonorrhoeae on
Johnson and Criss Neisseria gonorrhoeae and neutrophils
Frontiers in Microbiology | Cellular and Infection Microbiology April 2011 | Volume 2 | Article 77 | 10

Tapsall, J. W. (2009). Neisseria gonorrhoeae
and emerging resistance to extended
spectrum cephalosporins. Curr. Opin.
Infect. Dis. 22, 87–91.
Tjabringa, G. S., Vos, J. B., Olthuis, D.,
Ninaber, D. K., Rabe, K. F., Schalkwijk,
J., Hiemstra, P. S., and Zeeuwen, P. L.
(2005). Host defense effector mol-
ecules in mucosal secretions. FEMS
Immunol. Med. Microbiol. 45, 151–158.
Tramont, E. C., Ciak, J., Bosle go, J.,
Mcchesney, D. G., Brinton, C. C., and
Zollinger, W. (1980). Antigenic specifi-
city of antibodies in vaginal secretions
during infection with Neisseria gonor-
rhoeae. J. Infect. Dis. 142, 23–31.
Tseng, H. J., Mcewan, A. G., Apicella, M. A.,
and Jennings, M. P. (2003). OxyR acts
as a repressor of catalase expression in
Neisseria gonorrhoeae. Infect. Immun.
71, 550–556.
Tseng, H. J., Srikhanta , Y. , Mcewan,
A. G., and Jennings, M. P. (2001).
Accu mulat ion o f mang anese in
Neisseria gonorrhoeae correlates with
resistance to oxidative killing by
superoxide anion and is independent
of superoxide dismutase activity. Mol.
Microbiol. 40, 1175–1186.
Turner, S., Reid, E., Smith, H., and Cole,
J. (2003). A novel cytochrome c per-
oxidase from Neisseria gonorrhoeae:
a lipoprotein from a Gram-negative
bacterium. Biochem. J. 373, 865–873.
Tzeng, Y. L., Ambrose, K. D., Zughaier, S.,
Zhou, X., Miller, Y. K., Shafer, W. M.,
and Stephens, D. S. (2005). Cationic
antimicrobial peptide resistance in
Neisseria meningitidis. J. Bacteriol.
187, 5387–5396.
Tzeng, Y. L., Kahler, C. M., Zhang, X.,
and Stephens, D. S. (2008). MisR/
MisS two-component regulon in
Neisseria meningitidis. Infect. Immun.
76, 704–716.
Veal, W. L., Nicholas, R. A., and Shafer,
W. M. (2002). Overexpression of the
MtrC-MtrD-MtrE efflux pump due
to an mtrR mutation is required for
chromosomally mediated penicillin
resistance in Neisseria gonorrhoeae. J.
Bacteriol. 184, 5619–5624.
Veale, D. R., Finch, H., and Smith, H.
(197 6). Penetr ati on of penicillin
into human phagocytes containing
Neisseria gonorrhoeae: intracellular
surv ival and growth at optimum
concentrations of antibiotic. J. Gen.
Microbiol. 96, 353–363.
Veale, D. R., Goldner, M., Penn, C. W.,
Ward, J., and Smith, H. (1979). The
intracellular survival and growth of
gonococci in human phagocytes. J.
Gen. Microbiol. 113, 383–393.
Veale, D. R., Sen, D., Penn, C. W., Finch,
H., Smith, H., and Witt, K. (1977).
Interactions of Neisseria gonorrhoeae
with guinea pig defence mechanisms
family. Proc. Natl. Acad. Sci. U.S.A.
95, 1829–1833.
Siegel, M., Olsen, D., Critchlow, C., and
Buchanan, T. M. (1982). Gonococcal
pili: safety and immunogenicity in
humans and antibody function in
vitro. J. Infect. Dis. 145, 300–310.
Simons, M. P., Nauseef, W. M., and
Apicella, M. A. (2005). Interactions
of Neisseria gonorrhoeae with adher-
ent polymorphonuclear leukocytes.
Infect. Immun. 73, 1971–1977.
Simons, M. P., Nauseef, W. M., Griffith, T.
S., and Apicella, M. A. (2006). Neisseria
gonorrhoeae delays the onset of apop-
tosis in polymorphonuclear leuko-
cytes. Cell. Microbiol. 8, 1780–1790.
Skaar, E. P., Tobiason, D. M., Quick, J.,
Judd, R. C., Weissbach, H., Etienne,
F., Brot, N., and Seifert, H. S. (2002).
The outer membrane localization of
the Neisseria gonorrhoeae MsrA/B is
involved in survival against reactive
oxygen species. Proc. Natl. Acad. Sci.
U.S.A. 99, 10108–10113.
Soler-Garcia, A. A., and Jerse, A. E. (2004).
A Nei sse ria gon orrhoeae catalase
mutant is more sensitive to hydrogen
peroxide and paraquat, an inducer of
toxic oxygen radicals. Microb. Pathog.
37, 55–63.
Soler-Garcia, A. A., and Jerse, A. E. (2007).
Neisseria gonorrhoeae catalase is not
required for experimental genital tract
infection despite the induction of a
localized neutrophil response. Infect.
Immun. 75, 2225–2233.
Sorensen, O. E., Follin, P., Johnsen, A. H.,
Calafat, J., Tjabringa, G. S., Hiemstra, P.
S., and Borregaard, N. (2001). Human
cathelicidin, hCAP-18, is processed to
the antimicrobial peptide LL-37 by
extracellular cleavage with proteinase
3. Blood 97, 3951–3959.
St Amant, D. C., Valentin-Bon, I. E.,
and Jerse, A. E. (2002). Inhibition of
Neisseria gonorrhoeae by Lactobacillus
species that are commonly isolated
from the female genital tract. Infect.
Immun. 70, 7169–7171.
Stohl, E. A., Criss, A. K., and Seifert, H. S.
(2005). The transcriptome response
of Neisseria gonorrhoeae to hydrogen
peroxide reveals genes with previously
uncharacterized roles in oxidative
damage protection. Mol. Microbiol.
58, 520–532.
Stohl, E. A., and Seifert, H. S. (2006).
Neisseria gonorrhoeae DNA recom-
bination and repair enzymes protect
against oxidative damage caused by
hydrogen peroxide. J. Bacteriol. 188,
7645–7651.
Swanson, J., Barrera, O., Sola, J., and
Boslego, J. (1988). Expression of outer
membrane protein II by gonococci in
experimental gonorrhea. J. Exp. Med.
168, 2121–2129.
Sadarangani, M., Pollard, A. J., and Gray-
Owen, S. D. (2011). Opa proteins and
CEACAMs: pathways of immune
engagement for pathogenic Neisseria.
FEMS Microbiol. Rev. 35, 498–514.
Saigh, J. H., Sanders, C. C., and Sanders,
W. E. Jr. (1978). Inhibition of Neisseria
gonorrhoeae by aerobic and faculta-
tively anaerobic components of the
endocervical flora: evidence for a pro-
tective effect against infection. Infect.
Immun. 19, 704–710.
Sarafian, S. K., Tam, M. R., and Morse, S. A.
(1983). Gonococcal protein I-specific
opsonic IgG in normal human serum.
J. Infect. Dis. 148, 1025–1032.
Schook, P. O., Stohl, E. A., Criss, A. K.,
and Seifert, H. S. (2011). The DNA-
binding activity of the Neisseria gon-
orrhoeae LexA orthologue NG1427
is modulated by oxidation. Mol.
Microbiol. 79, 846–860.
Schwalbe, R. S., Sparling, P. F., and
Cannon, J. G. (1985). Variation of
Neis seria gonor rhoeae protein II
among isolates from an outbreak
caused by a single gonococcal strain.
Infect. Immun. 49, 250–252.
Seib, K. L., Simons, M. P., Wu, H. J.,
Mcewa n, A. G. , Nauseef, W. M.,
Apicella, M. A., and Jennings, M. P.
(2005). Investigation of oxidative
stress defenses of Neisseria gonorrhoeae
by using a human polymorphonu-
clear leukocyte survival assay. Infect.
Immun. 73, 5269–5272.
Seib, K. L., Wu, H. J., Kidd, S. P., Apicella,
M. A., Jennings, M. P., and Mcewan,
A. G. (2006). Defenses against oxida-
tive stress in Neisseria gonorrhoeae: a
system tailored for a challenging envi-
ronment. Microbiol. Mol. Biol. Rev. 70,
344–361.
Shafer, W. M. (1988). Lipopolysaccharide
masking of gonococcal outer-mem-
brane proteins modulates binding of
bacterial cathepsin G to gonococci. J.
Gen. Microbiol. 134, 539–545.
Shafer, W. M., and Morse, S. A. (1987).
Cleavage of the protein III and major
iron-regulated protein of Neisseria
gonorrhoeae by lysosomal cathepsin
G. J. Gen. Microbiol. 133, 155–162.
Shafer, W. M., Onunka, V. C., Jannoun,
M., and Huthwaite, L. W. (1990).
Molecular mechanism for the antigo-
nococcal action of lysosomal cathep-
sin G. Mol. Microbiol. 4, 1269–1277.
Shafer, W. M., Onunka, V. C., and Martin,
L. E. (1986). Antigonococcal activity of
human neutrophil cathepsin G. Infect.
Immun. 54, 184–188.
Shafer, W. M., Qu, X., Waring, A. J., and
Lehrer, R. I. (1998). Modulation of
Neisseria gonorrhoeae susceptibility
to vertebrate antibacterial peptides
due to a member of the resistance/
nodulation/division efflux pump
have undergone splenectomy. Clin.
Microbiol. Rev. 23, 740–780.
Ram, S., Mcquillen, D. P., Gulati, S., Elkins,
C., Pangburn, M. K., and Rice, P. A.
(1998a). Binding of complement fac-
tor H to loop 5 of porin protein 1A: a
molecular mechanism of serum resist-
ance of nonsialylated Neisseria gonor-
rhoeae. J. Exp. Med. 188, 671–680.
Ram, S., Sharma, A. K., Simpson, S. D.,
Gulati, S., Mcquillen, D. P., Pangburn,
M. K., and Rice, P. A. (1998b). A novel
sialic acid binding site on factor H
mediates serum resistance of sialylated
Neisseria gonorrhoeae. J. Exp. Med. 187,
743–752.
Ramsey, K. H., Schneider, H., Cross, A. S.,
Boslego, J. W., Hoover, D. L., Staley, T.
L., Kuschner, R. A., and Deal, C. D.
(1995). Inflammatory cytokines pro-
duced in response to experimental
human gonorrhea. J. Infect. Dis. 172,
186–191.
Rest, R. F. (1979). Killing of Neisseria
gonorrhoeae by human polymorpho-
nuclear neutrophil granule extracts.
Infect. Immun. 25, 574–579.
Rest, R. F., Fischer, S. H., Ingham, Z. Z.,
and Jones, J. F. (1982). Interactions
of Neisseria gonorrhoeae with human
neutrophils: effects of serum and
gonococcal opacity on phagocy te
killing and chemiluminescence. Infect.
Immun. 36, 737–744.
Rest, R. F., and Frangipane, J. V. (1992).
Grow th of Neiss er ia gon orrhoeae
in CMP-N-acetylneurami nic acid
inhibits nonopsonic (opacity-asso-
ciated outer m emb rane pro tei n-
mediated) interactions with human
neutrop hils . In fect . Immu n. 60 ,
989–997.
Rest , R. F., and Pretzer, E. (198 1).
Degr adat ion of gonococcal outer
membrane proteins by human neu-
trophil lysosomal proteases. Infect.
Immun. 34, 62–68.
Rice, P. A., and Kasper, D. L. (1982).
Characterization of serum resistance
of Neisseria gonorrhoeae that dissemi-
nate. Roles of blocking antibody and
gonococcal outer membrane proteins.
J. Clin. Invest. 70, 157–167.
Rock, J. P., and Rest, R. F. (1988). Rapid
damage to membranes of Neisseria
gonorrhoeae caused by human neu-
trop hil granule extr acts. J . Ge n.
Microbiol. 134, 509–519.
Roos, D., Van Bruggen, R., and Meischl, C.
(2003). Oxidative killing of microbes
by neutrophils. Microbes Infect. 5,
1307–1315.
Rouquette, C., Harmon, J. B., and Shafer,
W. M. (19 99) . Induct ion of the
mtrCDE-encoded efflux pump sys-
tem of Neisseria gonorrhoeae requires
MtrA, an AraC-like protein. Mol.
Microbiol. 33, 651–658.
Johnson and Criss Neisseria gonorrhoeae and neutrophils
www.frontiersin.org April 2011 | Volume 2 | Article 77 | 11

Jennings, M. P. (2005). Azurin of
pathogenic Neisseria spp. is involved
in defense against hydrogen peroxide
and survival within cervical epithelial
cells. Infect. Immun. 73, 8444–8448.
Wu, H. J., Seib, K. L., Srikhanta, Y. N.,
Kidd, S. P., Edwards, J. L., Maguire, T.
L., Grimmond, S. M., Apicella, M. A.,
Mcewan, A. G., and Jennings, M. P.
(2006). PerR controls Mn-dependent
resistance to oxidative stress in
Neisseria gonorrhoeae. Mol. Microbiol.
60, 401–416.
Zheng, H. Y., Alcorn, T. M., and Cohen, M.
S. (1994). Effects of H2O2-producing
lactobacilli on Neisseria gonorrhoeae
growth and catalase activity. J. Infect.
Dis. 170, 1209–1215.
Zughaier, S. M., Tzeng, Y. L., Zimmer,
S. M., Datta, A., Carlson, R. W., and
Stephens, D. S. (2004). Neisseria
meningitidis lipo oligosaccharide
structure-dependent activation of the
macrophage CD14/Toll-like receptor 4
pathway. Infect. Immun. 72, 371–380.
Confli ct of Interes t S tatement: T he
authors declare that the research was con-
ducted in the absence of any commercial
or financial relationships that could be
construed as a potential conflict of interest.
Received: 21 February 2011; paper pend-
ing published: 16 March 2011; accepted:
31 March 2011; published online: 13 April
2011.
Citation: Johnson MB and Criss AK (2011)
Resistance of Neisseria gonorrhoeae to
neutrophils. Front. Microbio. 2:77. doi:
10.3389/fmicb.2011.00077
This article was submitted to Frontiers in
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Witt, K., Veale, D. R., Finch, H., Penn,
C. W., Sen, D., and Smith, H. (1976).
Resistance of Neisseria gonorrhoeae
grown in vivo to ingestion and diges-
tion by phagocytes of human blood. J.
Gen. Microbiol. 96, 341–350.
Workowski , K. A., a nd Ber man, S.
(2010). Sexually transmitted diseases
treatment guidelines, 2010. MMWR
Recomm. Rep. 59, 1–110.
Wu, H., and Jerse, A. E. (2006). Alpha-2,3-
sialyltransferase enhances Neisseria
gonorrhoeae survival during experi-
mental murine genital tract infection.
Infect. Immun. 74, 4094–4103.
Wu, H., Soler-Garcia, A. A., and Jerse,
A. E. (2009). A strain-specific cata-
lase mutation and mutation of the
metal-binding transporter gene mntC
attenuate Neisseria gonorrhoeae in vivo
but not by increasing susceptibility to
oxidative killing by phagocytes. Infect.
Immun. 77, 1091–1102.
Wu, H. J., Seib, K. L., Edwards, J. L.,
Apicella, M. A., Mcewan, A. G., and
in subcutaneously implanted cham-
bers. FEMS Microbiol. Lett. 1, 3–6.
Virji, M. (2009). Pathogenic neisseriae:
surface modulation, pathogenesis and
infection control. Nat. Rev. Microbiol.
7, 274–286.
Virji, M., and Heckels, J. E. (1986). The
effect of protein II and pili on the inter-
action of Neisseria gonorrhoeae with
human polymorphonuclear leuco-
cytes. J. Gen. Microbiol. 132, 503–512.
Wain, B. S. (1947). The Unconquered
Plague. New York : Inter nat ional
University Press.
Warner, D. M., Shafer, W. M., and Jerse,
A. E. (2008). Clinically relevant muta-
tions that cause derepression of the
Neisseria gonorrhoeae MtrC-MtrD-
MtrE Efflux pump system confer dif-
ferent levels of antimicrobial resistance
and in vivo fitness. Mol. Microbiol. 70,
462–478.
Wiesner, P. J., and Thompson, S. E. III.
(1980). Gonococcal diseases. Dis. Mon.
26, 1–44.
Johnson and Criss Neisseria gonorrhoeae and neutrophils
Frontiers in Microbiology | Cellular and Infection Microbiology April 2011 | Volume 2 | Article 77 | 12