Examination of Neisseria gonorrhoeae opacity protein expression during experimental murine genital tract infection /

by Simms, Amy Nicole.

Title of Dissertation: Examination of Neisseria gonorrhoeae Opacity Protein Expression During Experimental Murine Genital Tract Infection Amy N. Simms, Doctor of Philosophy, 2005 Thesis directed by: Ann E. Jerse, Ph.D. Associate Professor, Department of Microbiology and Immunology The opacity (Opa) proteins of Neisseria gonorrhoeae are a family of phasevariable outer membrane proteins that bind to host cells. Phase variable expression occurs via a reversible frameshift mechanism within each opa gene. Opa protein expression is selected for, or induced during experimental genital tract infection of female mice, similar to that which was reported in male volunteers. Using a genetically marked strain of FA1090 to follow recovery of a specific population of Opa variants during murine infection, here we showed that selection of a pre-existing population of Opa-positive gonococci present in the inoculum was responsible for the reisolation of mainly Opa-positive variants early during infection. We conclude that the preferential iii recovery of Opa-positive gonococci observed early during murine infection is due to selection of a pre-existing population of Opa-positive variants caused by factors other than binding to human CEACAM receptors. In long-term infection of mice, a cyclical pattern of Opa protein expression was observed in which a decreased recovery of Opapositive variants followed early selection for Opa protein expression; reemergence of Opa-positive gonococci occurred later in infection. To examine rates of phase variation in a N. gonorrhoeae background under physiologically relevant conditions, we engineered a translational opaB::phoA fusion and introduced it into the chromosome of N. gonorrhoeae strain FA1090 to generate strain ANS100. No change was seen in the frequency or rate of opaB::phoA phase variation in all in vitro conditions tested. We next examined whether increased phase variation of the opaB::phoA fusion occurred during murine infection. No significant change occurred in the frequency of “on” variants among vaginal isolates when mice were inoculated with predominantly “off” variants; however, a marked increase in the recovery of “off” variants was observed following inoculation with predominantly “on” variants. The inability to show differences in opa gene phase variation under different conditions in vitro leads us to conclude at this time that induction of opa gene phase variation may be spontaneous and random. However, the in vivo studies suggest that increased opa gene phase variation may occur under conditions that we have not yet been able to mimic in vitro. iv To my parents, for everything v Examination of Neisseria gonorrhoeae Opacity Protein Expression During Experimental Murine Genital Tract Infection By Amy Nicole Simms Dissertation submitted to the Faculty of the Department of Microbiology and Immunology Graduate Program of the Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2005 vi