Pathogenesis and Clinical Manifestation of Neisseria gonorrhoeae

Neisseria gonorrhoeae is a gram-negative diplococcus that causes gonorrhea, a sexually transmitted infection that can affect the urethra, cervix, rectum, pharynx, and conjunctiva. Gonorrhea can lead to serious complications such as pelvic inflammatory disease, infertility, ectopic pregnancy, and disseminated gonococcal infection. The pathogenesis of Neisseria gonorrhoeae depends on its ability to evade the host immune system and to adhere to and invade the mucosal epithelial cells. To achieve this, the bacterium expresses various virulence factors or antigenic structures on its surface that mediate different aspects of infection. Some of the major virulence factors or antigenic structures of Neisseria gonorrhoeae are:

• Pili: These are hair-like appendages that extend from the bacterial surface and facilitate the exchange of genetic material between strains and the attachment to human mucosal cells. Pili also help the bacterium to invade host cells and to resist phagocytosis by neutrophils. Pili undergo genetic and phase variation to avoid recognition by host antibodies.
• Porin proteins (Por): These are proteins that form pores in the bacterial membrane and allow the entry of some nutrients. Por proteins also modulate the intracellular killing of gonococci by preventing the fusion of phagosomes and lysosomes within neutrophils. Moreover, Por proteins can bind to complement components C3b and C4b and confer variable resistance to serum bactericidal activity.
• Opacity proteins (Opa): These are proteins that function in the adhesion of gonococci within colonies and in the attachment of gonococci to host cell receptors such as heparin-related compounds and CD66 or carcinoembryonic antigen–related cell adhesion molecules. Opa proteins also induce endocytosis of gonococci by host cells and contribute to tissue tropism and immune evasion.
• Reduction modifiable protein (Rmp): This is a protein that associates with Por in the formation of pores in the cell surface. Rmp blocks the bactericidal effect of host IgG by interfering with the binding of antibodies to Por proteins.
• Lipooligosaccharide (LOS): This is a type of lipopolysaccharide that lacks long O-antigen side chains and is composed of lipid A, core oligosaccharide, and variable terminal structures. LOS is responsible for most of the toxicity and endotoxic effects of gonococcal infections, such as fever, inflammation, tissue damage, and shock. LOS also stimulates the release of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by host cells.
• Other proteins: These include Lip (H8), a heat-modifiable protein that is exposed on the bacterial surface; Fbp (ferric-binding protein), a protein that is expressed when iron supply is limited and helps the bacterium to acquire iron from host sources; and IgA1 protease, an enzyme that cleaves and inactivates IgA1, a major mucosal immunoglobulin of humans.

These virulence factors or antigenic structures enable Neisseria gonorrhoeae to colonize, invade, survive, and cause damage in the human host. Understanding their roles and mechanisms is essential for developing effective strategies for prevention, diagnosis, and treatment of gonorrhea.

Pili are hair-like appendages that extend up to several micrometers from the gonococcal surface. They are composed of multiple copies of a protein called pilin, which can vary in its amino acid sequence among different strains of Neisseria gonorrhoeae. This variation allows the bacteria to evade the host immune system by changing their antigenic structure.

Pili have two main functions in the pathogenesis of gonococcal infections: genetic exchange and attachment. Genetic exchange occurs when two gonococci come into contact and transfer DNA through their pili. This process, called transformation, can introduce new genes or alleles into the bacterial population, increasing their genetic diversity and adaptability. Some of the genes that can be transferred by transformation include those encoding for antibiotic resistance, pilin variation, and Opa proteins.

Attachment is the first step in the colonization of the human mucosal surfaces by gonococci. Pili mediate the initial interaction between the bacteria and the epithelial cells of the urethra, cervix, rectum, pharynx, and conjunctiva. Pili bind to specific receptors on the host cells, such as asialoglycoproteins and integrins, and trigger signaling pathways that lead to bacterial uptake and invasion. Pili also help the bacteria to resist phagocytosis by neutrophils, which are the main defense cells against gonococcal infections. Pili can interfere with the fusion of phagosomes and lysosomes, preventing the killing of the ingested bacteria.

Pili are therefore essential virulence factors for Neisseria gonorrhoeae, as they enable the bacteria to acquire new genetic traits, adhere to and invade the host cells, and evade the host immune response. Pili are also potential targets for vaccine development, as antibodies against pilin can block bacterial attachment and reduce infection rates. However, the high variability of pilin poses a challenge for designing an effective vaccine that can cover all possible strains of gonococci.

Porin proteins, also known as protein I (PI), are outer membrane proteins that extend through the gonococcal cell membrane and form pores in the surface through which some nutrients enter the cell . There are two main classes of porin proteins, PIA and PIB, that vary among different strains of gonococci and have different effects on the host immune response .

Porin proteins may impact intracellular killing of gonococci within neutrophils by preventing phagosome–lysosome fusion, a process that normally destroys the ingested bacteria. Porin proteins may also affect the resistance of gonococci to killing by normal human serum, which contains complement components that can lyse bacteria. Some porin proteins selectively bind to complement components C3b and C4b, which may interfere with the formation of the membrane attack complex that causes bacterial lysis.

Porin proteins are also involved in antigenic variation and immune evasion of gonococci. The genes encoding porin proteins undergo frequent recombination and mutation, resulting in changes in the amino acid sequence and expression level of the proteins. These changes may allow gonococci to escape recognition by host antibodies and immune cells, and to adapt to different microenvironments within the host.

Opacity proteins (Opa proteins) are surface-exposed proteins that are encoded by a family of 11 genes in Neisseria gonorrhoeae. These genes undergo frequent on-off switching, resulting in antigenic and phase variation of Opa expression. This allows the gonococcus to evade the host immune system and adapt to different host environments.

Opa proteins play a crucial role in the adhesion of gonococci within colonies and in the attachment of gonococci to host cell receptors. Opa proteins can bind to heparin sulfate proteoglycans (HSPGs), which are present on the surface of many human cells. HSPGs facilitate the initial adherence of gonococci to mucosal epithelial cells and also serve as co-receptors for other Opa-binding molecules.

One of the most important Opa-binding molecules is CD66 or carcinoembryonic antigen-related cell adhesion molecule (CEACAM). CEACAMs are members of the immunoglobulin superfamily that are expressed on various human cells, including epithelial cells, neutrophils, and B lymphocytes. CEACAMs mediate the tight binding and internalization of Opa-expressing gonococci into host cells. This process involves the activation of intracellular signaling pathways that modulate the cytoskeleton and membrane dynamics.

Opa proteins also interact with other host cell receptors, such as integrins and CD46, which may influence the invasion and survival of gonococci in different tissues. Moreover, Opa proteins can modulate the host immune response by inducing apoptosis of infected cells, inhibiting T cell proliferation, and suppressing cytokine production.

Opa proteins are therefore essential virulence factors of Neisseria gonorrhoeae that enable the pathogen to adhere, colonize, invade, and persist in various host niches. By varying their expression and antigenicity, Opa proteins also help the gonococcus to evade the host immune recognition and clearance.

Reduction modifiable protein (Rmp) is a surface-exposed protein that is antigenically conserved in all gonococci. It associates with Por in the formation of pores in the cell surface. Rmp has a unique property of changing its molecular weight from 35 kDa to 28 kDa when exposed to reducing agents such as dithiothreitol (DTT) or mercaptoethanol. This property is used to identify Rmp in immunoblots.

Rmp plays a crucial role in blocking the bactericidal effect of host IgG antibodies that are directed against other gonococcal antigens such as Por, Opa, and LOS. Rmp binds to the Fc region of IgG and prevents the activation of complement and the formation of membrane attack complex (MAC) on the gonococcal surface. This allows the gonococcus to evade the killing by serum and neutrophils.

Rmp also induces the production of non-bactericidal anti-Rmp IgG antibodies in the host, which further interfere with the bactericidal activity of other IgG antibodies. Anti-Rmp IgG antibodies can form immune complexes with Rmp and block its binding to bactericidal IgG antibodies. Alternatively, anti-Rmp IgG antibodies can bind to Rmp on the gonococcal surface and mask other antigens from recognition by bactericidal IgG antibodies.

The mechanism of Rmp-mediated immune evasion is illustrated in the following figure:

Figure: Rmp-mediated immune evasion. (A) Rmp binds to the Fc region of bactericidal IgG antibodies and prevents complement activation and MAC formation on the gonococcal surface. (B) Anti-Rmp IgG antibodies form immune complexes with Rmp and block its binding to bactericidal IgG antibodies. (C) Anti-Rmp IgG antibodies bind to Rmp on the gonococcal surface and mask other antigens from recognition by bactericidal IgG antibodies.

Rmp is a major virulence factor of Neisseria gonorrhoeae that contributes to its persistence and survival in the human host. It is also a potential target for vaccine development, as immunization with Rmp can elicit bactericidal antibodies that overcome the blocking effect of Rmp. However, Rmp is highly conserved among different strains of gonococci, which may limit its antigenic diversity and immunogenicity. Therefore, more studies are needed to explore the feasibility and efficacy of Rmp-based vaccines against gonorrhea.

Lipooligosaccharide (LOS) is a type of lipopolysaccharide (LPS) that lacks long O-antigen side chains. It is a major component of the outer membrane of Neisseria gonorrhoeae and plays a crucial role in its pathogenesis. LOS is composed of three parts: lipid A, core oligosaccharide, and variable extensions. The lipid A portion is responsible for the endotoxic activity of LOS, which can trigger inflammatory responses in the host. The core oligosaccharide is conserved among different strains of gonococci and contains phosphorylcholine, which can mimic host cell membrane components and facilitate attachment and invasion. The variable extensions are subject to antigenic variation and phase variation, which allow the gonococcus to evade the host immune system.

The toxicity of LOS in gonococcal infections is mainly due to its ability to stimulate the release of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-8 (IL-8), from various host cells, including macrophages, monocytes, endothelial cells, epithelial cells, and fibroblasts. These cytokines can cause fever, hypotension, shock, tissue damage, and increased vascular permeability. In addition, LOS can activate the complement system and induce the production of reactive oxygen species (ROS) and nitric oxide (NO), which can further damage the host cells and tissues.

The endotoxic effects of LOS are modulated by several factors, such as the dose, route, and duration of exposure, the presence of other bacterial components or host factors, and the genetic variation of both the gonococcus and the host. For example, some strains of gonococci can modify their lipid A structure by adding acyl groups or phosphate groups, which can reduce its endotoxicity. Some host factors, such as CD14, Toll-like receptor 4 (TLR4), and LPS-binding protein (LBP), can enhance or inhibit the recognition and signaling of LOS by the host cells. Some genetic polymorphisms in the host genes encoding these factors can also influence the susceptibility and severity of gonococcal infections.

The toxicity and endotoxic effects of LOS are important for the pathogenesis of Neisseria gonorrhoeae because they contribute to the local and systemic manifestations of gonorrhea, such as urethritis, cervicitis, pelvic inflammatory disease (PID), disseminated gonococcal infection (DGI), arthritis, endocarditis, meningitis, and septic shock. Moreover, they can also impair the mucosal barrier function and facilitate the transmission of other sexually transmitted infections (STIs), such as human immunodeficiency virus (HIV). Therefore, understanding the molecular mechanisms and regulation of LOS biosynthesis and expression in Neisseria gonorrhoeae may provide new insights into the prevention and treatment of gonorrhea and its complications.

Besides the proteins mentioned above, N. gonorrhoeae also expresses other surface proteins that contribute to its pathogenesis. These include:

• Lip (H8): This is a heat-modifiable protein that is exposed on the bacterial surface. It has a role in antigenic variation, as it can undergo phase variation by slipped-strand mispairing of a homopolymeric tract within its gene. It can also undergo antigenic variation by recombination with silent copies of the lip gene. The function of Lip in gonococcal infection is not fully understood, but it may be involved in adhesion, invasion, or immune evasion.
• Fbp (ferric-binding protein): This is a protein that binds iron and transports it across the outer membrane. It is expressed when the available iron supply is limited, such as in human infection. Iron is essential for the growth and survival of N. gonorrhoeae, as it is required for various metabolic processes and virulence factors. N. gonorrhoeae can acquire iron from human iron-binding proteins such as transferrin, lactoferrin, and hemoglobin by expressing specific receptors on its surface.
• IgA1 protease: This is an enzyme that cleaves and inactivates IgA1, a major mucosal immunoglobulin of humans. IgA1 protease helps N. gonorrhoeae to evade the mucosal immune response and to colonize the mucosal surfaces. It also modulates the inflammatory response by altering the cytokine production and chemotaxis of neutrophils.

These proteins are examples of how N. gonorrhoeae adapts to the host environment and evades the immune system by varying its antigenic structure and eliciting an inflammatory response.

Pathogenesis of Neisseria gonorrhoeae

Gonorrhoeal infection is generally limited to superficial mucosal surfaces lined with columnar epithelium . Pili and Opa proteins facilitate adhesion of the gonococcus to epithelial cells of the urethra, rectum, cervix, pharynx, and conjunctiva, thereby making colonization possible . Pili, PorB, and Opa proteins mediate gonococci to attach to mucosal cells, penetrate into the cells and multiply, and then pass through the cells into the subepithelial space where infection is established . The gonococcal LOS stimulates release of the proinflammatory cytokine tumor necrosis factor-α (TNF-α), which causes most of the symptoms associated with gonococcal disease . Antibodies to LOS can activate complement, releasing complement component C5a, which has a chemotactic effect on neutrophils; however, IgG and secretory IgA1 antibodies directed against Rmp protein can block this bactericidal antibody response . The gonococcus requires iron for growth and survival in vivo . The pathogen acquires this necessary nutrient by expression of specific transport systems that remove and internalize the iron from human iron binding proteins including transferrin, lactoferrin and hemoglobin .

The gonococcus is an incredibly versatile human pathogen that can cause a range of clinical manifestations depending on the site of infection and the host immune response. In women, gonococcal infection can result in cervicitis, urethritis, endometritis, salpingitis, pelvic inflammatory disease (PID), tubo-ovarian abscesses, ectopic pregnancy, infertility, and chronic pelvic pain . In men, gonococcal infection can cause urethritis, epididymitis, prostatitis, and infertility . Gonococcal infection can also affect extragenital sites such as the pharynx, rectum, eyes, joints, skin, heart valves, and central nervous system . Pharyngeal infection can be asymptomatic or cause sore throat and can facilitate transmission to other partners . Rectal infection can cause discharge, anal itching, soreness, bleeding, or painful bowel movements . Gonococcal conjunctivitis can cause purulent discharge, redness, swelling, and pain in the eyes and can lead to blindness if untreated . Gonococcal arthritis can cause fever, joint pain, swelling, and skin lesions . Gonococcal endocarditis can cause heart failure and embolic complications . Gonococcal meningitis can cause headache, stiff neck, fever, and altered mental status .

Gonorrhoea is a sexually transmitted disease that can have serious and permanent health consequences if left untreated. Early diagnosis and treatment are essential to prevent complications and transmission. However, the emergence of antimicrobial resistance in N. gonorrhoeae poses a major challenge for effective treatment and control of this infection .

One of the key steps in the pathogenesis of Neisseria gonorrhoeae is the adhesion and colonization of the gonococcus to the epithelial cells of the mucosal surfaces that it infects. This process involves several virulence factors that interact with host cell receptors and modulate the host immune response.

The most important virulence factor for adhesion is the type IV pilus (T4P), a hair-like appendage that extends from the bacterial surface and mediates attachment to human mucosal cells. The T4P is composed of a major subunit called PilE, which undergoes antigenic and phase variation to evade host immune recognition, and a minor subunit called PilC, which acts as a tip-associated adhesin and is essential for pilus extension. PilC binds to host cell receptors such as CD46, a complement regulatory protein, and asialoglycoprotein receptor (ASGP-R), a lectin-like receptor. PilC also induces endocytosis of the gonococcus into epithelial cells, where it can survive and replicate.

Another important virulence factor for adhesion is PorB, a porin protein that forms pores in the bacterial outer membrane and allows nutrient uptake. PorB also binds to host cell receptors such as Toll-like receptor 2 (TLR2), a pattern recognition receptor that activates inflammatory signaling pathways, and CD66 or carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), a family of immunoglobulin-like molecules that are involved in cell-cell interactions. PorB modulates the host immune response by inducing apoptosis of epithelial cells, inhibiting phagosome-lysosome fusion in neutrophils, and interfering with complement-mediated killing.

A third important virulence factor for adhesion is Opa, an opacity protein that is expressed on the bacterial surface and undergoes antigenic and phase variation. Opa proteins function in adhesion of gonococci within colonies and in attachment of gonococci to host cell receptors such as heparan sulfate proteoglycans (HSPGs), which are present on the basement membrane and extracellular matrix, and CEACAMs, which are also expressed on epithelial cells, neutrophils, and natural killer cells. Opa proteins also facilitate invasion of epithelial cells by inducing membrane ruffling and actin rearrangement.

By expressing these virulence factors, N. gonorrhoeae can adhere to and colonize various epithelial tissues of the urogenital tract, rectum, pharynx, and conjunctiva, thereby establishing infection and causing disease.

One of the major virulence factors of Neisseria gonorrhoeae is its lipooligosaccharide (LOS), which is a truncated form of lipopolysaccharide (LPS) that lacks the O-antigen side chains. LOS is exposed on the outer membrane of the gonococcus and can interact with host cells and immune system components. LOS has both toxic and endotoxic effects that contribute to the pathogenesis and symptoms of gonococcal infections.

One of the key effects of LOS is the stimulation of the release of proinflammatory cytokine tumor necrosis factor-α (TNF-α) from host cells, especially macrophages and epithelial cells. TNF-α is a potent mediator of inflammation and immune responses that can have both beneficial and detrimental effects on the host. TNF-α can activate other immune cells, such as neutrophils, to enhance phagocytosis and killing of bacteria. TNF-α can also induce the expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), on endothelial cells and epithelial cells, which facilitate the recruitment and migration of leukocytes to the site of infection. TNF-α can also increase the permeability of blood vessels, allowing plasma proteins and fluid to leak into the tissues and cause edema and tissue damage.

However, excessive or prolonged production of TNF-α can also have harmful effects on the host. TNF-α can induce apoptosis or programmed cell death in some cell types, such as epithelial cells, which can compromise the integrity of the mucosal barrier and increase susceptibility to secondary infections. TNF-α can also cause systemic effects, such as fever, hypotension, shock, and organ failure, if it enters the bloodstream in high concentrations. TNF-α can also aggravate autoimmune diseases, such as rheumatoid arthritis and inflammatory bowel disease, by enhancing inflammation and tissue destruction.

The stimulation of TNF-α release by gonococcal LOS is mediated by several mechanisms. One mechanism is through the binding of LOS to Toll-like receptor 4 (TLR4), which is a pattern recognition receptor that recognizes LPS and other microbial components. TLR4 is expressed on various cell types, including macrophages and epithelial cells. Upon binding to LOS, TLR4 activates a signaling pathway that involves several adaptor proteins, such as MyD88 and TRIF, and leads to the activation of transcription factors, such as nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3), which induce the expression of genes encoding TNF-α and other inflammatory cytokines.

Another mechanism is through the binding of LOS to CD14, which is a co-receptor for TLR4 that enhances its signaling. CD14 is expressed on macrophages and monocytes, but also exists in a soluble form in plasma and other body fluids. Soluble CD14 can bind to LOS and transfer it to TLR4 on cell surfaces, thereby amplifying the inflammatory response.

A third mechanism is through the binding of LOS to complement receptors, such as CR3 and CR4, which are expressed on neutrophils and macrophages. Complement receptors recognize complement components that are attached to bacterial surfaces as a result of complement activation by antibodies or alternative pathways. Complement receptors can trigger phagocytosis and oxidative burst in neutrophils and macrophages, but can also induce TNF-α production through intracellular signaling pathways.

The role of gonococcal LOS in stimulating TNF-α release is important for understanding the pathogenesis and clinical manifestation of gonorrhea. TNF-α is responsible for most of the signs and symptoms associated with gonococcal disease, such as urethritis, cervicitis, proctitis, pharyngitis, conjunctivitis, arthritis, endocarditis, meningitis, and disseminated gonococcal infection (DGI). TNF-α also plays a role in modulating the immune response to gonococcal infection, either by enhancing or suppressing protective immunity. Therefore, targeting TNF-α or its signaling pathways may be a potential strategy for preventing or treating gonorrhea.

Iron is an essential nutrient for many biological processes, such as respiration, DNA synthesis, and enzyme activity. However, iron is also toxic at high concentrations, and therefore its availability and homeostasis are tightly regulated by the host and the pathogen. Neisseria gonorrhoeae, like other pathogenic Neisseria species, has evolved a repertoire of high-affinity iron acquisition systems to facilitate essential iron uptake in the human host .

Acquisition of iron by N. gonorrhoeae requires both the energy-harnessing cytoplasmic membrane protein TonB and specific outer membrane TonB-dependent transporters (TdTs). TdTs are proteins that bind and extract iron from human iron-binding proteins, such as transferrin, lactoferrin, hemoglobin, and hemoglobin-haptoglobin complexes . N. gonorrhoeae can also use siderophores, which are small iron-chelating molecules produced by other bacteria or fungi, as iron sources. N. gonorrhoeae expresses TdTs for the siderophores enterobactin, salmochelin, aerobactin, and ferrichrome.

The expression of TdTs is regulated by the availability of iron and other environmental factors, such as oxygen, pH, and temperature. N. gonorrhoeae uses a transcriptional regulator called Fur (ferric uptake regulator) to sense the intracellular iron concentration and control the expression of TdTs and other genes involved in iron metabolism. Fur also regulates the expression of genes involved in virulence, such as LOS biosynthesis, pili production, and Opa proteins.

The ability to acquire iron from different sources is crucial for N. gonorrhoeae to colonize and infect different mucosal sites in the human body. Iron acquisition also influences the interaction of N. gonorrhoeae with the host immune system, as some TdTs can modulate complement activation, cytokine production, and neutrophil function. Therefore, understanding the mechanisms of iron acquisition by N. gonorrhoeae may provide insights into its pathogenesis and potential targets for intervention.

Neisseria gonorrhoeae infection can cause a variety of clinical manifestations depending on the site of infection, the host immune response, and the presence of complications. The most common sites of infection are the urogenital tract, the rectum, the pharynx, and the conjunctiva. The infection can also disseminate to other parts of the body, causing skin lesions, arthritis, endocarditis, or meningitis.

• Urogenital infection: The most common symptom of gonorrhea in males is urethritis, which presents as dysuria and purulent urethral discharge. The incubation period ranges from 2 to 14 days, with a median of 4 days. In females, cervical infection is often asymptomatic or causes mild symptoms such as vaginal discharge, dysuria, or intermenstrual bleeding. The incubation period is less well defined in females than males. Urogenital infection can ascend to cause complications such as epididymitis in males and pelvic inflammatory disease (PID) in females. PID can result in tubal scarring, infertility, ectopic pregnancy, and chronic pelvic pain .
• Rectal infection: Rectal infection can occur in both males and females who engage in receptive anal intercourse. It can also occur by autoinoculation from genital secretions. Rectal infection is often asymptomatic or causes mild symptoms such as anal discharge, pruritus, or pain. In some cases, it can cause proctitis with tenesmus, rectal bleeding, and mucopurulent discharge .
• Pharyngeal infection: Pharyngeal infection can occur in both males and females who engage in oral sex. It is usually asymptomatic or causes nonspecific symptoms such as sore throat or pharyngeal erythema. Pharyngeal infection can rarely cause pharyngeal abscess or systemic dissemination .
• Conjunctival infection: Conjunctival infection can occur in both adults and neonates by direct contact with infected secretions. In adults, it usually results from autoinoculation from genital or pharyngeal sites. In neonates, it results from vertical transmission during delivery. Conjunctival infection causes purulent conjunctivitis with eyelid edema, chemosis, and discharge. It can lead to complications such as corneal ulceration, perforation, or blindness .
• Disseminated gonococcal infection (DGI): DGI occurs when gonococci spread through the bloodstream to distant sites. It affects about 0.5 to 3 percent of patients with gonorrhea. DGI can present as a triad of skin lesions (pustules or hemorrhagic macules), arthritis (monoarticular or polyarticular), and tenosynovitis (especially of the wrists and ankles). Other manifestations include fever, malaise, myalgia, and arthralgia. DGI can also cause rare but serious complications such as endocarditis, meningitis, osteomyelitis, or hepatitis.

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