Gonococcal

''Neisseria gonorrhoeae'', also known as ''gonococcus'' (singular) or ''gonococci'' (plural), is a species of Gram-negative diplococci bacteria first isolated by Albert Neisser in 1879. An obligate human pathogen, it primarily colonizes the mucosal lining of the urogenital tract; however, it is also capable of adhering to the mucosa of the nose, pharynx, rectum, and conjunctiva. It causes the sexually transmitted genitourinary infection gonorrhea as well as other forms of gonococcal disease including disseminated gonococcemia, septic arthritis, and gonococcal ophthalmia neonatorum.

''N. gonorrhoeae'' is oxidase positive and a microaerophile that is capable of surviving phagocytosis and growing inside neutrophils. Culturing it requires carbon dioxide supplementation and enriched agar (chocolate agar) with various antibiotics ( Thayer–Martin). It exhibits antigenic variation through genetic recombination of its pili and surface proteins that interact with the immune system.

Sexual transmission is through vaginal, anal, or oral sex. Sexual transmission may be prevented through the use of barrier protection. Perinatal transmission may occur during childbirth, though it is preventable through antibiotic treatment of the mother before birth and application of antibiotic eye gel on the eyes of the newborn. Gonococcal infections do not result in protective immunity; therefore, individuals may be infected multiple times. Reinfection is possible due to ''N. gonorrhoeae's'' ability to evade the immune system by varying its surface proteins.

Asymptomatic infection is common in both males and females. Untreated infection may spread to the rest of the body (disseminated gonorrhea infection), especially the joints (septic arthritis). Untreated infection in women may cause pelvic inflammatory disease and possible infertility due to the resulting scarring. Gonorrhoea is diagnosed through cultures, Gram staining, or nucleic acid tests (i.e. polymerase chain reaction) of urine samples, urethral swabs, or cervical swabs. Chlamydia co-testing and testing for other STIs is recommended due to high rates of co-infection.

Antibiotic resistance in ''N. gonorrhoeae'' is a growing public health concern, especially given its propensity to develop resistance easily. This ability of ''N. gonorrhoeae'' to rapidly adapt to novel antimicrobial treatments has been seen several times since the 1930s, making numerous treatment plans obsolete. Some strains have exhibited resistance to the current ceftriaxone treatments.

Microbiology

''Neisseria'' species are fastidious, Gram-negative cocci (though some species are rod-shaped and occur in pairs or short chains) that require nutrient supplementation to grow in laboratory cultures. They are facultative intracellular pathogens, meaning they are able to persist and colonize within host cells but can also multiply outside the host cellular environment. They typically appear in pairs (diplococci), resembling the shape of coffee beans. Members of this genus do not form endospores and are nonmotile, with the exception of pathogenic species, which capable of moving using twitching motility; most are also obligate aerobes. Of the 17 species that colonize humans, only two are pathogenic: ''N. gonorrhoeae,'' which causes gonorrhea, and '' N. meningitidis'', a leading cause of bacterial meningitis.

Culture and identification

''N. gonorrhoeae'' can be isolated on Thayer–Martin agar (or VPN) agar in an atmosphere enriched with 3-7% carbon dioxide. Thayer–Martin agar is a chocolate agar plate (heated blood agar) containing nutrients and antimicrobials (vancomycin, colistin, nystatin, and trimethoprim). This agar preparation facilitates the growth of ''Neisseria'' species while inhibiting the growth of contaminating bacteria and fungi. Martin Lewis and New York City agar are other types of selective chocolate agar commonly used for ''Neisseria'' growth. ''N. gonorrhoeae'' is oxidase positive (possessing cytochrome c oxidase) and catalase positive (able to convert hydrogen peroxide to oxygen). When incubated with the carbohydrates lactose, maltose, sucrose, and glucose, ''N. gonorrhoeae'' will oxidize only the glucose.

Metabolism

Carbon

Unlike other ''Neisseria'' species that can also metabolize maltose, ''N. gonorrhoeae'' is capable of using only glucose, pyruvate, and lactate as central carbon sources, and glucose is catabolized via both the Entner-Doudoroff (ED) and pentose phosphate (PP) pathways, and the ED pathway is the primary oxidative method. Use of these pathways is necessary as ''N. gonorrhoeae'' is incapable of glucose catabolism via the Embden-Meyerhof-Parnas (EMP) pathway due its lack of the phosphofructokinase (PFK) gene; however, the fructose 1,6-bisphosphatase enzyme is present to allow for gluconeogenesis to occur.

Glucose is first metabolized through the ED pathway to produce pyruvate and glyceraldehyde 3-phosphate, the latter of which can then further metabolized by enzymes of the EMP pathway to yield another molecule of pyruvate. The resultant pyruvate molecules are then converted into acetyl-CoA, which can then be incorporated as a substrate for the citric acid cycle (CAC) to yield high-energy electron carriers that will be used by the electron transport chain (ETC) for ATP production; however, the CAC is largely used for generating biosynthetic precursors rather than for catabolic purposes. This is due in part to inhibited expression of several CAC enzymes in the presence of glucose, pyruvate, or lactate. These enzymes, namely citrate synthase, aconitase, and isocitrate dehydrogenase, are needed for the incorporation of acetate. Instead, a partial CAC has been observed, where α-ketoglutarate is formed by glutamate dehydrogenase or transamination of oxaloacetate and glutamate by aspartate aminotransferase (yielding aspartate and α-ketoglutarate). The CAC then continues from there to yield oxaloacetate, which is an important precursor molecule for a number of biosynthetic pathways. Another differentiating aspect of the gonococcal CAC is the lack of malate dehydrogenase, which is instead replaced by a membrane-bound malate:quinone-oxidoreductase that operates independently of NAD⁺ by directly transferring electrons to ubiquinone.

Conversely, acetyl-CoA that does not enter the CAC but enters the phosphotransacetylase- acetate kinase (PTA-AckA) pathway, where it can be converted into acetate by phosphorylation (to form acetyl phosphate and release coenzyme A) and dephosphorylation to form ATP. While this acetate can enter the CAC for further oxidation, this does not occur so long as other carbon sources such as glucose or lactate are present, in which case it is excreted from the cell or incorporated for lipid synthesis. ''N. gonorrhoeae'' lack the glyoxylate shunt, preventing them from using acetate to form CAC intermediates to replenish the cycle.

A significant portion of the glyceraldehyde 3-phosphate formed in gonococci is recycled via the gluconeogenic pathway to reform glucose 6-phosphate, as well as the intermediate fructose 6-phosphate. Both of these can then be used for pentose synthesis in the PP pathway via the oxidative and non-oxidative pathways, respectively, for subsequent nucleotide formation as well as energy production.

''N. gonorrhoeae'', like other pathogenic members of the genus ''Neisseria'', are capnophiles, meaning they require higher-than-normal concentrations of carbon dioxide (CO₂) to grow, either in the form of CO₂ or bicarbonate (HCO₃⁻) depending on the bacterial strain. This requirement must be met exogenously during the lag and stationary growth phases, though it appears to be met through high metabolic CO₂ productions in the exponential phase. Assimilation of this CO₂ in ''Neisseria'' species is done by carbonic anhydrase and phosphoenolpyruvate enzymes in the periplasmic space and the cytoplasm, respectively.

Lactate catabolism is also of particular importance for gonococci, both for pathogenicity and for growth. External lactate is transported in to the cell via lactate permease (LctP). The ''N. gonorrhoeae'' genome encodes for three lactate dehydrogenase (LDH) enzymes for that allow for metabolism of both ''L''-lactate and ''D''-lactate: a cytoplasmic NAD⁺-dependent ''D''-lactate dehydrogenase (LdhA), which is responsible for and two membrane-bound LDHs, one specific to ''L''-lactate (LldD) and the other specific to ''D''-lactate (LdhD). The membrane-bound LDHs have been determined to be flavoprotein-containing respiratory enzymes that directly oxidize lactate to reduce ubiquinone. While these enzymes do not directly pump protons (H⁺ ions) into the periplasmic space, it is proposed that the reduction of ubiquinone by these enzymes is capable of feeding into the larger ETC.

Electron transport chain and oxidative phosphorylation

As an obligate human pathogen and a facultative anaerobic capnophile, ''Neisseria gonorrhoeae'' typically colonize mucosal surfaces in microaerobic environments, such as those in the genitourinary tract. Growth in areas where oxygen concentrations are limited requires a terminal oxidase with a high affinity for oxygen; in gonococci, oxygen reduction is performed by a ''ccb₃'' -type cytochrome oxidase. In addition to aerobic respiration, gonococci can also perform anaerobic respiration via the reduction of nitrite (NO₂) to nitric oxide (NO) as well as reduction of NO to nitrous oxide (N₂O).

There are several enzymes that contribute electrons to the intramembranous ubiquinone pool, the first step in the ETC. These include the membrane bound LDHs (LldD and LdhD), NADH:ubiquinone oxidoreductase (aka NADH dehydrogenase; Nuo complex I), Na⁺-translocating NADH dehydrogenase (Nqr), succinate dehydrogenase (SDH), and the membrane-bound NAD⁺-independent malate:quinone-oxidoreductase (MqR).

Following the initial transfer of electrons to ubiquinone, proposed schematics for the organization of the gonococcal ETC suggest the electrons can be further passed down the chain by reduction of the cytochrome ''bc₁'' complex or can be directly transferred to NO as a terminal electron acceptor by NO reductase (NorB). In the case of the former, electrons can then be passed from the ''bc₁'' complex along two alternative pathways via the reduction of either cytochrome ''c₄'' or ''c₅''. Both of these cytochromes transfer electrons to the terminal cytochrome ''ccb₃'' oxidase for the reduction of O₂ to form H₂O under aerobic conditions.

Gonococci are also reduce NO₂ via an inducible outer membrane-attached copper-containing nitrite reductase (AniA, a member of the NirK protein family) under anaerobic conditions, though this process has also been noted in microaerobic conditions as a means of supplementing growth. This leads to the formation of NO that is subsequently reduced to N₂O in a partial denitrification pathway. The ''ccb₃'' oxidase of ''N. gonorrhoeae'', dissimilarly to other members of the ''Neisseria'' genus, is a tri-heme protein that can transfer electrons not only to O₂ (conserved across ''Neisseria'' species) but also to AniA for NO₂ reduction. This is in addition to the typical process of receiving electrons transferred from cytochrome ''c₅''.

The general purpose of the ETC is the formation of the electrochemical gradient of hydrogen ions (H⁺ or protons), resulting from concentration differences across the plasma membrane, needed to power ATP production in a process known as oxidative phosphorylation. In gonococci, movement of protons into the periplasmic space is accomplished by the Nuo complex I, the cytochrome ''bc₁'' complex, and cytochrome ''ccb₃''. Subsequently, ATP synthesis is performed by the F₁F₀ ATP synthase, a two-part protein complex present in gonococci as well as numerous other species across phylogenetic domains. This complex couples proton translocation back into the cytoplasm along its gradient with mechanical rotation to generate ATP.

Iron

The general purpose of the ETC is the formation of the electrochemical gradient of hydrogen ions (H⁺ or protons), resulting from concentration differences across the plasma membrane, needed to power ATP production in a process known as oxidative phosphorylation. In gonococci, movement of protons into the periplasmic space is accomplished by the Nuo complex I, the cytochrome ''bc₁'' complex, and cytochrome ''ccb₃''. Subsequently, ATP synthesis is performed by the F₁F₀ ATP synthase, a two-part protein complex present in gonococci as well as numerous other species across phylogenetic domains. This complex couples proton translocation back into the cytoplasm along its gradient with mechanical rotation to generate ATP.

To acquire the necessary iron, gonococci produce TonB-dependent transporters (TDTs) on the surface of their outer membrane that are able to directly extract iron, along with other metals, from their respective carrier proteins. Some of these include transferrin binding proteins A (TbpA) and B (TbpB), lactoferrin-binding proteins A (LbpA) and B (LbpB), and hemoglobin/hemoglobin-haptoglobin binding proteins HpuB and HpuA. In addition to these proteins, gonococci are also capable of using siderophores, or compounds that are capable of chelating iron in the environment, that are produced by other bacteria; however, gonococcal cells are incapable of synthesizing siderophores themselves. These xenosiderophores are taken up by the TDT FetA through the outer membrane and then brought into the cell by the ''fetBCDEF'' transporter system.

Along with the sequestration defence that can be further upregulated by host inflammation, humans also produce siderocalins that are able to chelate siderophores to as a further method of inhibiting pathogenic bacterial growth. These are sometimes ineffective against ''N. gonorrhoeae'', which is able to colonize intracellularly, particularly in phagocytic cells such as macrophages and neutrophils. Increases in host intracellular iron also down regulates some of the intracellular pathogen-killing mechanisms; coincidentally, pathogenic ''Neisseria'' are able to alter several host cell mechanisms that ultimately allow the pathogen to take most of the available iron away from the host immune cell.

Surface molecules

On its surface, ''N. gonorrhoeae'' bears hair-like pili, surface proteins with various functions, and sugars called lipooligosaccharide. The pili mediate adherence, movement, and DNA exchange. The opacity-associated (Opa) proteins interact with the immune system, as do the porins. Lipooligosaccharide is an endotoxin that provokes an immune response. All of these are antigenic and exhibit antigenic variation. The pili, Opa proteins, porins, and even the lipooligosaccharide have mechanisms to inhibit the immune response, making asymptomatic infection possible.

Opa proteins

Phase-variable opacity-associated (Opa) adhesin proteins are used by ''N. gonorrhoeae'' as part of evading immune response in a host cell. At least 12 Opa proteins are known and the many variations of surface proteins make recognizing ''N. gonorrhoeae'' and mounting a defense by immune cells more difficult. Opa proteins are in the outer membrane and facilitate a response when the bacteria interacts with a variety of host cells. These proteins bind to various epithelial cells, and allow ''N. gonorrhoeae'' to increase the length of infection as well as increase the amount of invasion into other host cells.

Type IV pili

Dynamic polymeric protein filaments called type IV pili allow ''N. gonorrhoeae'' to do many bacterial processes including adhesion to surfaces, transformation competence, twitching motility, and immune response evasions. To enter the host the bacteria uses the pili to adhere to and penetrate mucosal surfaces. The pili are a pivotal virulence factor for ''N. gonorrhoeae''; without them, the bacterium is unable to promote colonization. For motility, individual bacteria use their pili in a manner that resembles a grappling hook: first, they are extended from the cell surface and attach to a substrate. Subsequent pilus retraction drags the cell forward. The resulting movement is referred to as twitching motility. ''N. gonorrhoeae'' is able to pull 100,000 times its own weight, and the pili used to do so are amongst the strongest biological motors known to date, exerting one nanonewton. The PilF and PilT ATPase proteins are responsible for powering the extension and retraction of the type IV pilus, respectively. The adhesive functions of the gonococcal pilus play a role in microcolony