VIRULANCE FACTORS

N.gonorrhoeaeANIMAL AND CELL CULTURE MODELS. N.gonorrhoeae is a human-specific pathogen. Thus, in vivo studies are usually done with human volunteers. You might think that few volunteers would come forward, but the experience is actually not all that bad. Only male volunteers are used, and they are inoculated in the urethra (a painless procedure if an uncomfortable one for many). And watched closely for several days to determine whether the strain being tested will cause disease. Once signs of the disease are evident, the volunteer is given an antibiotic that clears up the infection in short order. And of course , human volunteers are paid or given some other reward.

N.gonorrhoeae adheres to and invades many different types of tissue culture cell. A popular cell line (because it is an epithelial cell line) is Chang conjunctival epithelial cells, but epithelial cell lines derived from cervical and prostate carcinomas have also been widely used. N.gonorrhoeae will not adhere to and invade nonhuman cell lines such as CHO (Chinese hamster ovary) cells, unless the cells have been transfected with cloned human DNA so that they are producing the receptor found on human cells. Such cell lines can be useful for testing individual receptors one at a time, whereas human cells may have multiple receptors.

PMNs and macrophagelike cell lines are used to study the fate of N.gonorrhoeae ingested by phagocytes. There is an animal model of sorts. Mice implanted subcutaneously with a chamber into which the bacteria are injected are used as a model for studying virulence factors associated with systemic spread. Fallopian tissue provides an organ culture model for studying effects of bacterial colonization of fallopian tubes. This type of tissue is readily available in any hospital where hysterectomies are done.

N.gonorrhoeae is naturally transformable (takes up DNA without chemical shock or electroporation ), but the transformation system discriminates against foreign DNA. There are transposons that integrate in the N.gonorrhoeae chromosome (e.g., Tn916 and its derivatives), but integration is not completely random. Tn1545del, a derivative of a conjugative transposon which is closely related to Tn916, has been used succesfully in some transposon mutagenesis experiments but, like Tn916, probably doesnot integrate completely randomly. Gene distruption and gene replacement are possible but note as easy as with E. Coli. The genetic tools are getting better all the time, however.

ADHERENCE AND INVASION . The picture of how gonorrhea progresses has changed significantly in recent years. N.gonorrhoeae was once classified as an extracellular pathogen., which was only found outside cells, except for PMNs. Today, invasion and intracellular residence are believed to be an important part of the gonococcal lifestyle. The first stage in the infection process is colonization of a mucosal surface lined by columnal epithelial cells. As a result of the inflammatory response elicited by the bacteria, the bacteria

soon encounter phagocytes, especially PMNs. PMNs in the purulent discharge of a person with gonorrhea often contain numerous bacteria.

There is some uncertainty about whether the bacteria inside phagocytes are in the process of being killed or whether N. gonorrhoeae can survice inside PMNs. Microscopic analysis of bacteria inside PMNs indicates that many are in the process of being broken down and killed, but a fractioan of the ingested bacteria appear to remain viable. Whatever the intracellular fate of N. gonorrhoeae inside PMNs, it appears that N. gonorrhoeae, like many of the bacteria described in previous chapters, can also bind to human epithelial cells and stimulate these cells to phagocytose them by a process that involves actin rearrangement. A major gonococcal adhesion is type 4 pili; its pilin subunits are processed by a type II secretion system (see Appendix 1). The first amino acid in the mature protein is a modified phenylalanine. This is the same type of pilus found on a number of human and plant pathogens. The receptor for the N. gonorrhoeae pilus is a human cell surface protein called CD46. CD46 is normally involved in regulation of the complement cascade.

The gonococci produce a number of variants of the type 4 pili. Some variants from bundles, and these bundle-forming pili appear to attach more efficiently to host cells than pili that do not form bundles. The reason some pili from bundles has not been established with certainty but pilin proteins are glycosylated. One explanation is that lake of glycosylation makes the pili more able to stick together, forming the bundles. This introduces a theme that is seen repeatedly in gonococcal virulence factors: the variability of surface antigens. The finding that bacterial proteins could be glycosylated was a surprise. Prior to this discovery, the dogma had been that eukaryotic cells glycosylated proteins but prokaryotic cells did not.

After adhering to the surface of cervical or urethral epithelial cels, the bacteria are ingested by the epithelial celland transcytose through the cell to the basolateral surface, where they exit into the space below the cells. This signals the body to mount the pronounced inflammatory response that responsible for most of the symptoms of gonorrhea. This progression can be demonstrated in cervical tissue samples (ex vivo organ culture). There is some contoversy as to whether binding via pili is sufficient to induce phagocytosis of the bacteria or whether some other intermediate is required. Gonococcal outer membrane porin proteins (called PorA and PorB) may have a role in triggering phagocytosis. First, both PorA and PorB proteins can nucleate actin. Thus, they could contribute to the actin rearrengements that contribute to formation of the pseudopods that engulf the bacteria. Second, PorA and PorB proteins can enter the cytoplasmic membranes of eukaryotic cells, causing transient changes in membrane potential. This could be a signal that sets off the signal transduction cascade that leads to ingestion of the bacteria.

Another type of surface protein, which was originally thought to have a role in adherence to the host cell but may actually have another role in virulence, is Opafection proteins. Opa proteins are a family of gonococcal outer membrane proteins. The name opa stands for opacity; bacteria with opa proteins on their surfaces form colonies that have a more

opaque-looking surface than those not expressing the protein. This easily visible phenotype has been used to identify mutantas that express aberrant levels of opas. The reason opa proteins were once thought to be adhesins was that bacteria producing opa but not pili were taken up by tissue culture cells. Two findings have raised questions about the involvement of opa proteins in the early steps of infection. First, bacteria that produce pili but not opa proteins are taken up by epithelial cells in organ cultures and transcytose similarly to wild-type bacteria. Thus, opa proteins are not essential for uptake. A second problem is that the receptors for opa proteins are located on the basolateral surface of properly polarized cells. Opa proteins seem to be important for virulence, because mutants lacking them are less virulent in human volunteers than wild-type bacteria, but their precise role in the progression of infection is still uncertain. Some of them mediate opsonin-independent uptake of the bacteria by PMNs. If the bacteria are able to survive in PMNs, a characteristic that is still in dispute, opa proteins might be aimed more at PMNs than at epithelial cells.