Gonococci – Pathogens of Growing Importance. Part 2. Virulence Factors, Antimicrobial Resistance and Vaccine Development

N. gonorrhoeae adhesins recognize different target sites on host cells, allowing the pathogen to interact with various tissues during gonorrheal infection. Adhesion and invasion into human cells are a crucial step in developing infection. Antigenic and phase variability of adhesins protects bacteria from effective immune defense. An important virulence factor is the glycolipid outer membrane antigen LOS, composed of endotoxinactive lipid A and a core oligosaccharide. It stimulates the release of pro-inflammatory cytokines, which promotes neutrophil recruitment to the infection site (Ng et al. 2005; Quillin and Seiferth 2018). The structure of LOS lacks the repeated polysaccharide chains characteristic of the analogous cell wall antigen of Enterobacterales, lipopolysaccharide. The branched LOS molecule has three oligosaccharide chains anchored to the cell membrane by lipid A. The variable chains are attached via a 3-deoxy-D-manno-octulosonic acid molecule to two heptose residues of lipid A: Hep1 and Hep2. The oligosaccharide core shows considerable variability, even within the same N. gonorrhoeae strain due to phase variation in numerous glycosyl transferase genes. This enzyme, which is crucial in the synthesis of LOS, is responsible for attaching further sugars to the oligosaccharide chain. The variability of LOS is also due to the genetic diversity of alleles encoding glycosyltransferases (Apicella et al. 1987; Banerjee et al. 1998; Shafer et al. 2002). LOS is involved in the adherence process to epithelial cells. Variability and modifications of LOS structure, such as attachment of exogenous sialic acid molecules, promote evasion of host immune defense mechanisms. Both sialylation of gonococcal LOS and attachment of phosphoethanolamine to lipid A protect against antibodies and reduce susceptibility to bacterial killing by neutrophils, inhibit complement activation, and increase resistance to antimicrobial peptides (Lewis et al. 2009; Balthazar et al. 2011). The importance of LOS in processes leading to oviduct damage has also been described (Gregg et al. 1981; Cooper et al. 1986).

TFPs are protein filamentous surface structures anchored to the cytoplasmic membrane, passing through the outer membrane to the outside of the cell. As adhesins, they have a crucial function in the pathogenesis of gonorrheal infection. Their presence is related to the virulence of the strain. They participate in the initial phase of bacterial adhesion to human epithelial cells and interactions with neutrophils. Pili, binding to the CD46 receptor, initiates adherence to eukaryotic cells. Based on the principle of twitching motility, they provide gonococci with a degree of motility, which promotes the spread of microorganisms across the epithelial surface and facilitates their auto-aggregation. Phase variation in TFP expression enables gonococci to defend against phagocytosis and evade the host immune response. Several genes encode different subunits of pili. The main subunit of the pilE protein shows antigenic variation due to recombination with several silent copies at the pilS loci. Pili are also important in the natural transformation of N. gonorrhoeae (Chen and Dubnau 2004).

Opa proteins are a family of 24–30 kDa outer membrane proteins. As adhesins, they are involved in gonococcal auto-aggregation and receptor-mediated adherence of bacteria to eukaryotic cells and in stimulation and modification of the immune response. Opa proteins bind to host cell CEACAM receptors (carcinoembryonic antigen family adhesion molecules). They are named after a phenomenon observed macroscopically, as they cause the lack of transparency of colonies cultured on clear agar. Depending on the expression of Opa proteins, the morphology of gonococci colonies on agar varies. Opa colonies expressing Opa + are referred to as “opaque phenotype.” Based on differences in binding to two types of cell receptors, two classes of Opa proteins have been distinguished, grouping 11 different types. Opa proteins class one (Opa₅₀) bind to the heparan sulfate proteoglycan receptor on epithelial cells using heparan sulfate. Opa class two (Opa_51–60) binds to CD66 antigen family molecules on epithelial cells, lymphocytes and neutrophils, i.e., the receptor molecules CEACAM-1, 3, 5 and 6. The ability of bacteria to interact with different types of receptors during infection activates distinct signal transduction pathways in cells and is an essential adaptive feature (Gray-Owen et al. 1997; Chen et al. 1997). Opa proteins increase bacterial resistance to complement action. Opa protein expression is subject to antigenic and phase variation. There can be up to 11 different opa genes in the genome of N. gonorrhoeae strain, with several types being expressed simultaneously. The protein sequence of all Opa proteins is 70% identical. Conservative regions of the molecule are not exposed on the surface of the outer membrane, in contrast to the highly variable outer fragments, the second and third loops. These regions are called hypervariable domains 1 and 2 (Stern et al. 1986; Bhat et al. 1992).

PorB is the most abundant outer membrane protein of N. gonorrhoeae, encoded by the porB gene. It is essential for gonococci viability as a conserved voltage-gated ion channel-like protein. It consists of three protein subunits. Each monomer, 32–35 kDa, has a structure of β-folded barrel, 16 trans-membrane segments, and eight characteristic extracellular loops with high variability (Chen and Seifert 2013). In gonococci, there are two different Porin B isoforms – PorB1a and PorB1b-, resulting from stable expression of one of the two alleles of the porB gene in a given bacterial strain. Due to this diversity, two distinct phenotypes of gonococci can be distinguished depending on the type of porin they possess: P. IA and P. IB. Strains with the P. IA phenotype are characterized by increased invasiveness, which has been found based on epidemiological data describing the more frequent involvement of this particular phenotype in generalized gonococcal infections (Bash et al. 2005, Guglielmino et al. 2022). The P. IA phenotype strains are characterized by the deletion of a fragment in loop 5, which gives them resistance to trypsin digestion and higher resistance to the complement-dependent lethal effects of human serum (Blake et al. 1981; Ram et al. 2001). P. IB phenotype gonococci are more often isolated from genitourinary tract infections and usually show sensitivity to the bactericidal effect of serum. The nucleotide sequences of both types of porins show about 80% similarity. Genetic variation within PorB distinguishes strains in epidemiological studies (Fudyk et al. 1999; Liao et al. 2009). These porins are the basis for serotyping based on reactions with monoclonal antibodies (Sandström et al. 1980, Tam et al. 1982). A fragment of the third outer loop of the PorB molecule has become, along with the TbpB fragment, the basis of NG-MAST typing (Martin et al. 2004), and the PorB1b gene segment encoding 30 amino acids is a well-characterized determinant of antibiotic resistance, referred to as penB (Olesky et al. 2002; Unemo et al. 2011). PorB acts as a mitogen, activates B lymphocytes and stimulates cytokine production. On the other hand, this protein influences the phagocytosis process, enabling it to block complement activation through the alternative or classical pathway and protecting bacteria from opsonization. Factor H, the main regulator of the alternative complement pathway, binds directly to PorB1a. Strains with the P. IB phenotype require LOS sialylation to bind factor H. Serum resistance in gonococci is also mediated by the C4bp protein binding to different fragments – extracellular loop 1 within PorB1a of P. IA strains and loops 5 and 7 of PorB1b (P. IB). PorB can also inhibit oxidative burst and neutrophil apoptosis, facilitating pathogens to survive infection despite the inflammatory response (Chen et al. 2011; Chen and Seifert 2013; Palmer and Criss 2018).

Rmp is an outer membrane protein physically associated with PorB, presenting the potential of an immunology evasion (Joiner et al. 1985). As a highly conserved membrane protein, it stimulates the synthesis of “blocking antibodies” specific for both LOS and PorB, inhibiting serum’s bactericidal effect (Gulati et al. 2015). The presence of antibodies to Opa proteins and the absence of “blocking antibodies” induced by Rmp was associated with the reduction of upper reproductive tract infection in high-risk women (Plummer et al. 1993a, 1994b).

PG fragments that are spontaneously released during the growth and division of bacterial cells at the site of infection are important virulence factors. Recognized by human cytosolic nucleotide-binding oligomerization domains 1 and 2, pattern recognition receptors are an element that induces the inflammatory response (Mavrogiorgos et al. 2014). By stimulating the inflammatory process, they are responsible for, among others, damage within the fallopian tubes and destruction of the epithelial cells. The release of pro-inflammatory peptidoglycan monomers as well as dimers and free peptides, leading to remodelling of the cell wall structure of gonococci, is possible due to the activity of bacterial enzymes, mainly lytic transglycosylases (Cloud et al. 2002), as well as amidases and endopeptidases (Schaub et al. 2019).

OMVs by N. gonorrhoeae (Pettit et al. 1992) are characteristic of many pathogenic Gam-negative bacteria (Kulp et al. 2010). OMVs were first described in Vibrio cholerae in 1967 and recognized as artefacts of in vitro liquid medium culture (Chatterjee SN, Das J, 1967). Vesicles are formed by the protrusion of the bacterial outer membrane, the detachment of a fragment of this membrane and the formation of a spherical, closed structure with a diameter of 20–200 nm. Inside OMVs, components of both the bacterial periplasm and cytoplasm, such as adhesins, enzymes, and DNA fragments, can be encapsulated. Examination of gonococcal OMVs proteins concentration can suggest active sorting of proteins during natural blebbing of bacteria facilitating vesicles functions (Zielke et al. 2014; Deo et al. 2018). So far, these potent single lipid bilayer spheres, carrying plenty of outer membrane lipids, proteins, LOS, periplasmatic PG fragments and proteins, cytoplasmatic proteins and nucleic acids, have shown to be the promise vaccine antigens (van der Pol et al., 2015). OMVs contain many outer membrane antigens, presented in their native form, as derived straightly from the crucial pathogen adhesion and immunogenicity surface layer of Gram-negative bacterial cell wall. Polyantigenic gonococcal OMVs can potentially overcome the challenges of high phase and antigen variation of N. gonorrhoeae.

An important phenomenon in the pathogenesis of gonorrhoea is the ability to utilize the host’s iron resources with the help of gonococcal membrane transporters. Unlike most bacteria, Neisseria species pathogenic to humans do not produce siderophores and use iron bound to human glycoproteins, mainly transferrin (West and Sparling 1985). They can also obtain iron from lactoferrin (Mickelsen and Sparling 1981; Mickelsen et al. 1982), hemoglobin and heme, which are available in the female reproductive tract during menstruation. Eight iron transporters dependent on TonB proteins of the inner membrane of gonococci have been described: TbpA/TbpB, LbpA/ LbpB, HpuB/ HpuA, FetA, TdfF, TdfG, TdfH, TdfJ, which enable efficient utilization of protein-bound iron (Cornelissen and Hollander 2011). Human transferrin is found in the highest concentrations in serum, cerebrospinal fluid and joint fluid. Still, it can also be detected in semen and mucous membranes, especially in inflamed tissue. Iron acquisition requires energy and direct contact between the iron transport glycoprotein and the bacterial cell surface. Increased expression of gonococcal genes (e.g., tbp, lbp, fbp) responsible for iron uptake from transferrin and lactoferrin has been found during gonococcal infection of the lower genital tract (McClure et al. 2015).

Active efflux-type membrane transporters are an essential group of surface proteins that act as pumps that remove harmful chemicals from the bacterial cell. The expression level of such efflux transporters determines the variable level of sensitivity of the microorganism to antimicrobial substances naturally present in their environment and to antibiotics. Gonococcal membrane transporters belong to different families of bacterial pumps. The FarA-FarB transporter of the Major Facilitator System (MFS) family recognizes antibacterial long-chain fatty acids (Lee and Shafer 1999). The NorM transporter is a member of the Multidrug and Toxic Compound Extrusion (MATE) family, and its overexpression can reduce the sensitivity of gonococci to ciprofloxacin and norfloxacin (Rouquette-Loughlin et al. 2005). The MtrC-MtrD-MtrE system belongs to the Resistance-Nodulation-Cel Division (RND) family of pumps found in Gram-negative bacteria and depends on energy drawn from ATP. The MtrCDE pump is an active transporter of substances from the cytoplasm and periplasmic space (Maness and Sparling 1973). It can remove hydrophobic compounds, naturally occurring antimicrobial peptides, bile salts, progesterone, detergent and dye-like compounds, and antibiotics (Delahay et al. 1997). The mtrCDE pump comprises three protein subunits, C, D, and E, passing through the outer membrane, periplasmic space, and inner membrane. It has a typical operon, and the MtrR repressor and MtrA activator regulate its expression. The mtrR repressor gene is an important determinant of gonococcal chromosomal antibiotic resistance. As a result of point mutations within the mtrR promoter or the actual gene, or due to the mosaic structure of mtrR resulting from HGT derived from related species of the genus Neisseria, there is increased expression of a pump that removes toxic substances from the bacterial cell. Phenotypically, this increases MICs for macrolide antibiotics, tetracyclines, penicillin and cephalosporins (Zarantonelli et al. 2001).

Anaerobic metabolism in N. gonorrhoeae is enabled by two enzymes of the denitrification pathway: coppercontaining nitrite reductase (AniA) that reduces nitrite to nitric oxide (NO) and nitric oxide reductase (NorB) (Barth et al. 2009). The NorB enzyme may be involved in removing toxic NO produced by macrophages. The expression of these enzymes is strictly regulated by oxygen availability, and elevated expression of AniA and NorB was found, e.g., in biofilms (Falsetta et al. 2009).

The increasing antimicrobial resistance of N. gonorrhoeae is a global problem, and it has a complex basis, both chromosomal and plasmid-mediated (Fig. 1) (Unemo et al. 2016). Features of gonococci responsible for the rise of antibiotic resistance include genetic plasticity, the ability to transform naturally, high levels of antigenic and phase variation, point mutations, mosaicism of chromosomal determinants of resistance, and plasmids with resistance-determinant genes. A structurally diverse group of β-lactamase plasmids encoding blaTEM penicillinases determines penicillin resistance. In contrast, conjugative plasmids may have Tet(M) tetracycline resistance genes in their structure (Fig. 2) (Pachulec et al. 2014). Based on phenotypic characteristics, strains with plasmid resistance to penicillin and tetracycline, respectively, can be identified using the cephinase test for penicillinase-producing isolates (PPNG or penicillinase-producing N. gonorrhoeae) and a MIC value for tetracycline ≥ 16.0 mg/L, characterizing strains with high levels of tetracycline resistance – the High-Level tetracycline resistant (HLTR) phenotype (Muhammad et al. 2014).

Fig. 1.

Fig. 2.

Strains exhibiting low-level tetracycline resistance (LLTR), phenotypically expressed by tetracycline MIC values in the range of 1–8 mg/l, do not have the plasmid tet(M) gene, and the resistance in their case is chromosomal and may be the result of changes in genes associated with drug susceptibility, among others, in the mtrR gene – encoding the membrane pump repressor MtrCDE or in the porB1b (penB) gene (Pitt et al. 2019). An example of chromosomal loci transfers in N. gonorrhoeae, including important determinants of antibiotic resistance, is the mosaic structure of PBP2 (penA) proteins associated with resistance to penicillin and cephalosporins (Muhammad et al. 2002; Nakayama et al. 2016) and the mosaic structure of mtrR and mtrCDE genes, encoding the pump gene repressor and membrane pump MtrCDE, respectively (Rouquette-Loughlin et al. 2018). Single nucleotide mutations that change the target site of antibiotic action in a bacterial cell cause resistance to penicillin, cephalosporins, macrolides, fluoroquinolones, and spectinomycin. Mutations within the genes of penicillin-binding protein PBP1 (ponA) cause an increase in the MIC for penicillin, while within the genes of PBP2 (penA) for penicillin and third-generation cephalosporins (Lindberg et al. 2007). Point mutations, e.g., adenine deletion (35Adel) in the promoter region and substitutions of the coding region in the mtrR gene (A39T and G45D), can result in overexpression of the MtrCDE pump and lead to increased resistance to penicillin, tetracyclines, macrolides and cephalosporins (Zarantonelli et al. 2001). Mutations in the porB1b gene fragment, also referred to as a penB resistance determinant, altering the structure of the PorB1 membrane protein can result in reduced membrane permeability and impede the entry of tetracyclines, penicillin and cephalosporins into the cell. The main PorB1b sequence changes described so far result in amino acid substitutions of G120 and A121 (Lindberg et al. 2007). Resistance to azithromycin is mainly caused by mutations in the 23SrRNA subunit allele, reducing the affinity of macrolide for the 50S ribosome unit. Mutations within the DNA gyrase (gyrA) and topoisomerase IV (parC) genes cause resistance to fluoroquinolones. Mutations in ribosomal genes encoding the 16sRNA unit and the 5S protein, rpsE, inhibit spectinomycin binding to the ribosome (Unemo et al. 2016).

A bacterial biofilm is understood as a spatial structure within which bacterial cells adhere to a surface, contact each other, cooperate and are surrounded by a matrix. The extracellular polymeric mainly comprises bacterial products, glycoproteins, lipids, and nucleic acids. The matrix’s biochemical nature and the biofilm’s structure depend on the microorganisms’ properties and environmental conditions (Stoodley et al. 2002; Sauer et al. 2003). Bacterial biofilm can form on the surface of living cells and is natural for many commensal microorganisms colonizing human mucous membranes. At the same time, the biofilm phenomenon is important in terms of the pathogenicity of microorganisms. After entering the body in planktonic form, bacterial pathogens can adhere to host cells and then gradually form a biofilm. It has been found that bacterial cells residing in biofilms are characterized by high viability and increased resistance to unfavorable environmental conditions, such as physical and chemical changes in the environment (e.g., nutrient deficiency, changes in pH or oxygen concentration), and better tolerate the presence of bactericidal substances. Growth in the form of a biofilm facilitates long-term colonization of a variety of tissues and enhances bacterial resistance to natural antimicrobial peptides, other immune system factors, and antibiotics (Donlan and Costerton 2002).

The biofilm formed by N. gonorrhoeae was visualized in cervical biopsy preparations (Steichen et al. 2008). A three-dimensional 48-hour biofilm of the reference strain N. gonorrhoeae 1291 formed on transformed cervical epithelial cells was imaged by confocal microscopy (Steichen et al. 2008). The ability of gonococci to form a biofilm on the abiotic surface under conditions of continuous flow of medium and on the surface of urethral and cervical epithelial cells in ex vivo cultures was described (Greiner et al. 2005), according to the work of Falsetta et al. The transcriptomes of gonococci growing in the biofilm differ from those of planktonic cells, as demonstrated using the RNA microarray technique. Biofilms showed a significant increase in the expression of genes related to the metabolism of anaerobic respiration AniA, NorB and the cytochrome C peroxidase (ccp) gene associated with oxidative stress tolerance (Falsetta 2009).

Many mechanisms of avoiding the host immune system protection have been discussed while describing gonococci’s main virulence factors. N. gonorrhoeae is both an extra- and intracellular pathogen able to induce a strong inflammatory response through the Th-17 pathway but a weak, insufficient adaptive response. The innate immune response is a first line of defence against gonococci. Lack of protective immunity after recovery, asymptomatic infections in women and long-term complications of gonorrhoea indicate that the pathogen possesses the mechanisms of immunological evasions (Płaczkiewicz, 2019).

The ability of gonococci to evade immune defense mechanisms, including oxidative burst and killing by neutrophil granulocytes, should be underlined. Generally, neutrophils are the first phagocytic defense line in bacterial infections and represent the major component of the inflammatory response in gonococcal infection. The influx of numerous activated polymorphonuclear leukocytes (PMNs) into the urethra following gonococcal emergence on epithelium is, in fact, typical of symptomatic infection in men (Rest and Shafer 1989). The inflammatory response in symptomatic gonorrhoea is stimulated by gonococcal cell membrane adhesins, peptidoglycan fragments released naturally during cell division, A lipid of LOS, surface lipoproteins, heptoses, which are intermediates of LOS biosynthesis, and methylated DNA fragments secreted by strains possessing a type IV secretion system (Palmer and Criss 2018). Neutrophils kill microorganisms through the activity of antimicrobial proteins and synthesize ROS (Segal et al. 2005). However, despite the presence of numerous neutrophils in the urethra during N. gonorrhoeae infection, live gonococci are cultured from the purulent secretions collected from the patient, showing that PMNs are ineffective in killing N. gonorrhoeae (Rest and Shafer 1989). As gonococci, during infection, are exposed to different sources of reactive oxygen species (ROS), they use many factors to protect themselves from oxidative damage: catalase, cytochrome c oxidase, methionine peptide sulfoxide reductase, cytochrome c peroxidase, bacterioferritin, manganese uptake system (Criss et al. 2021). The IgA protease, produced and secreted by the gonococci, capable of degrading the hinge site of IgA class immunoglobulins present on human mucous membranes, is also a virulence factor helping evasion of host epithelial immunity control (Quillin and Seiferth 2018).

Host immune response to gonococci has not been fully understood. Another interesting issue is how the reproductive tract microbiota influences human susceptibility to gonococcal infections. Due to the vaginal epithelium colonization by microorganisms, the totality of which is referred to as vaginal microbiota (VM) interaction of immune cells, epithelial cells, commensals and pathogens in this niche are complex. In a physiological state, the domination of Lactobacillus spp. on the vaginal mucosa, with much less amounts of other microorganisms, including Gardnerella, Bifidobacterium, Streptococcus, Ureaplasma, Corynebacterium, Enterococcus etc. are observed. Lactobacillus spp. is responsible for maintaining stability and a healthy vaginal environment (Ravel et al. 2011). Gardnerella vaginalis is a component of the vaginal microbiota in healthy women and also the dominant microorganism in the vagina in a state of bacterial vaginosis. The reproductive tract microbiota associations with susceptibility to infection by sexually transmitted pathogens have been shown (Brotman et al. 2010).