For the efficient colonization of human mucosal epithelial cells, N. gonorrhoeae has evolved dedicated surface components, including, pili, colony opacity (Opa) proteins, and lipooligosaccharide (LOS). All three components are well-studied virulence factors of these bacteria and undergo phase and/or antigenic variation, allowing gonococci to evade the human immune response (Fig.1.2).
Fig. 1.2: Major virulence factors contributing to gonococcal colonization of epithelial cells. The outer membrane (OM) of gonococci contains the filamentous pili, the integral Opa proteins and LOS. All three virulence factors are associated with host cell attachment and are important for the establishment and maintenance of a gonococcal infection (modified from Virji 2009).
1.3.1 Pili
The initial attachment of gonococci to the apical side of epithelial tissues is mediated by the type IV pili. Hence, studies with human male volunteers demonstrated that the pili is crucial for the establishment of a successful infection of the urethra (Swanson et al. 1987). The pilus fibre is composed of numerous PilE subunits (pilin), which are arranged in a helical configuration.
Additionally, several minor pilus subunits (PilC, PilV and PilX) can be
incorporated into the fiber and modulate its function. Among them, PilC is thought to be the major pilus adhesin. Thus, it was shown that PilC mutants do not adhere to epithelial cells (Rudel et al. 1992).
Pili undergo antigenic variation via several recombinase A-dependent homologous recombination events (Haas and Meyer 1986). Beside the expression locus pilE, which contains the functional pilin gene, a number of silent, promotor-less pilS genes are encoded on the gonococcal chromosome.
Variation of the pilE gene arises from recombination between pilS copies and the pilE locus, generating mosaics of the pilE gene and different numbers of pilS storage copies. In addition, the pilus varies through phase variation of the pilC genes, regulated by insertion or deletion of single nucleotides into a homopolymeric run of G within the coding sequence (Jonsson et al. 1991).
Although, the gonococcal pilus is well studied, its corresponding receptor on human epithelial cells remains elusive. Former investigations identified the transmembrane glycoprotein CD46 as pilus receptor, however no direct interaction of CD46 and PilC has been observed (Kallstrom et al. 1997).
Additionally, further studies could not confirm an essential role of CD46 in pilus-mediated cell adherence (Kirchner et al. 2005).
1.3.2 Lipooligosaccharide (LOS)
Gonococcal LOS is composed of a highly hydrophobic lipid A and an attached hydrophilic nonrepeating oligosaccharide chain. In contrast to the common lipopolysaccharide (LPS) of other Gram-negative bacteria, gonococcal LOS lacks the repeating O-carbohydrate antigen side chain. Variation of gonococcal LOS is mediated by phase variation of enzymes employed in the biosynthesis of LOS (Shafer et al. 2002).
LOS is also thought to be involved in gonococcal cell adherence. Accordingly, it was shown that LOS with a terminal lacto-N-neotetraose group interacts with the asialoglycoprotein receptor expressed on primary urethral epithelial cells (Harvey et al. 2001). In addition, it was demonstrated, that LOS lipid A recruits the complement molecule C3b. Inactive C3b (iC3b), together with gonococcal pili and porin promote complement receptor type 3 (CR3) binding, which allows gonococci to colonize and invade cervical epithelial cells (Edwards and Apicella 2002; Edwards et al. 2002).
1.3.3 Opa proteins
Opa proteins contribute, as the name already indicates, to the opaque phenotype of gonococcal colonies on solid growth media. In total, up to twelve different Opa proteins can be expressed in gonococci. Opa proteins form a β-barrel secondary structure, integrated in the outer membrane of gonococci.
They are composed of eight antiparallel β-strands and four extracellular loops.
Two of these loops are highly variable in their amino acid sequence (HV1 and HV2), one loop is semivariable (SV) and one loop is conserved among different Opa variants. In contrast to the pil loci, the opa gene loci are complete genes, which are constitutively transcribed. However similar to pili and LOS, Opa proteins undergo phase variation (Stern et al. 1986). This is due to a pentameric repeat sequence (CTCTT) within the 5’ coding region of each opa gene. This sequence is variable in length and depending on the number of repeat units the opa gene is in frame and expressed in full-length or owing to a premature stopcodon a truncated non-functional Opa protein version is expressed. The coding repeat variation is based on a RecA-independent DNA slipped strand mispairing mechanism during DNA replication. Phase variation of individual opa genes occurs independently at a frequency of 10-3, resulting in heterogeneous gonococcal populations where single bacteria express none, one, or multiple different Opa proteins. Due to the fact that it is not possible to control, which Opa protein variants are expressed in a gonococcal population, it is not easy to study the function of single Opa proteins. Furthermore, Opa proteins are highly diverse among different gonococcal strains. Thus, a former study revealed that 14 unrelated gonococcal strains had no opa alleles in common (Bilek et al.
2009). Novel gonococcal opa genes arise mainly by homologous recombination events between opa loci of the same chromosome, as well as between different organisms. Thereby, novel alleles are mostly generated by shuffling of already existing HV regions of distinct opa genes. In contrast, gene duplication and de novo amino acid mutations occur much less frequently than changes due to homologous recombinations of different HV regions (Bilek et al. 2009). The diversity of gonococcal Opa proteins is most likely due to the selective pressure of the human immune system and their importance for gonococcal colonization.
Gonococcal Opa proteins play, beside pili, a central role in the establishment and the maintenance of a gonococcal infection. After the initial pili-mediated
attachment, Opa proteins are responsible for a more intimate interaction of gonococci with epithelial cells. The importance of Opa proteins during a gonococcal infection was underscored by a study which showed that the gonococci isolated from urethra of male volunteers, who were previously challenged with piliated, negative gonococci, were predominantly Opa-positive (Swanson et al. 1988; Jerse et al. 1994). This experiment provides evidence that there is a strong in vivo selection for the opaque phenotype of gonococci in their natural host.
Gonococcal Opa proteins are known to target human cell surface receptors.
Thus, it was shown that gonococci expressing Opa50 interact with heperan-sulphate proteoglycans (HSPGs) expressed on Chang conjunctiva epithelial cells. Interaction of Opa50 and HSPGs finally results in bacterial internalization (Chen et al. 1995; van Putten and Paul 1995). This internalization is dependent on the activation of the phosphatidylcholine-dependent phospholipase C (PC-PLC9), which generates the second messenger diaglycerol (DAG), which in turn activates the acidic sphingomyelinase (ASM). ASM generates ceramide that seems to be involved in actincytoskeleton reorganization and bacterial uptake (Esen et al. 2001). In addition, Opa50-expressing gonococci bind via vitronectin or fibronectin to integrin αvβ5 orα5β1, respectively (Duensing and van Putten 1997; Gomez-Duarte et al. 1997; van Putten et al. 1998). Simultaneous interaction of Opa50 with HSPGs and integrins again triggers gonococcal uptake by epithelial cells.