Mechanisms of Neisseria Gonorrhoeae Survival in Primary Human Neutrophils
Johnson, Morgan, Microbiology - Graduate School of Arts and Sciences, University of Virginia
Criss, Alison, Department of Microbiology, University of Virginia
Neisseria gonorrhoeae (gonococcus, Gc) is the causative agent of the sexually transmitted disease gonorrhea which affects 106 million people each year worldwide. There is currently no protective vaccine against Gc, and due to the rise of antibiotic resistance strains, there is increased necessity for new treatment options. Infection with Gc results in a potent neutrophil (polymorphonuclear leukocyte, PMN)-driven inflammatory response which creates the purulent exudate, a hallmark of gonorrheal infection. PMNs store antimicrobial peptides and proteases in cytoplasmic granules. Upon PMN activation these granules fuse at the plasma or phagosome membrane to release their contents extracellularly or intracellularly, respectively, in order to kill microorganisms. Gc is quite remarkable because despite the variety of mechanisms used by PMNs to kill microbes, viable Gc can be cultured from human gonorrhea exudates and are found intracellularly in PMNs infected ex vivo. These data indicate that Gc has evolved mechanisms to avoid and defend against PMN mediated killing. In this thesis I investigated the mechanisms of Gc intracellular survival in PMNs and determined how one Gc surface structure, opacity-associated (Opa) proteins, affects Gc survival inside PMNs.
Gc has 11 opa genes, encoding for 7-9 distinct Opa proteins. As a result of phase variation during gonorrheal infection, there is a heterogeneous population of Gc expressing no, one, or multiple Opa proteins. In order to determine how Opa protein expression affects Gc survival in PMNs, we utilized a Gc strain with in frame deletions of all 11 opa genes (Opa- Gc) or a strain constitutively expressing the OpaD protein (Opa+ Gc). Consistent with previous observations, we observed that Opa+ Gc have reduced survival in PMNs compared to Opa- Gc. Characterization of the survival defect demonstrated that Opa+ Gc have significantly reduced intracellular survival compared to Opa- Gc. PMNs kill bacteria intracellularly by fusing cytoplasmic granules with bacteria containing phagosomes in order to expose bacteria to oxidative and non-oxidative antimicrobial products. Our data demonstrates the novel finding that Opa+ Gc reside in mature phagosomes, while Opa- Gc phagosomes delay fusion with PMN primary granules. We observed that viable Gc were more likely to reside in immature phagosomes. Additionally, increasing primary granule fusion with Opa- Gc phagosomes was sufficient to decrease intracellular survival, and likewise decreasing primary granule fusion with Opa+ Gc phagosomes was sufficient to increase intracellular survival. In agreement with previous reports, our results indicate that even in the presence of reactive oxygen species, PMN mediated killing of Gc is primarily non-oxidative, specifically due to the activity of phagosomal serine proteases. Opa+ and Opa- Gc display similar sensitivity to PMN non-oxidative antimicrobial components, indicating differences in intrinsic sensitivity to antimicrobial components does not account for the Opa+ intracellular survival defect. From these data we conclude that avoidance of PMN primary granule fusion with Gc phagosomes is a central mechanism used by Gc to survive inside human PMNs.
PHD (Doctor of Philosophy)
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