Neisseria Gonorrhoeae Sialylates its Lipooligosaccharide to Subvert the Human Neutrophil Response

Cardenas, Amaris, Microbiology - School of Medicine, University of Virginia
Criss, Alison, MD-MICR Microbiology, University of Virginia

Neisseria gonorrhoeae (Gc) is the causative agent of gonorrhea, a sexually transmitted infection that when left untreated, can lead to pelvic inflammatory disease, ectopic pregnancy, and infertility. The damage to the reproductive tract is a direct result of the potent immune response mounted by the human host, which is characterized by an influx of neutrophils to infection sites. Despite this effort, Gc has evolved to develop various mechanisms to resist killing by the human immune system, enabling the bacteria to persist in highly inflammatory environments and for viable Gc to be recovered from purulent urethral exudates. Gc can manipulate its interactions with neutrophils by modifying its surface lipooligosaccharide (LOS), including sialylation.
Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-NANA) scavenged from the host using LOS sialyltransferase (Lst), as Gc cannot produce its own sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In this thesis, I elucidated a mechanism by which Gc exploits host-derived sialic acid to counteract neutrophil antigonococcal activity in complement-independent manner.
To test how sialylation affects Gc-neutrophil interactions, I used an infection model with adherent, interleukin 8-treated primary human neutrophils. Neutrophils were exposed to Gc of strain FA1090 constitutively expressing a single Opacity associated protein (Opa). Opa proteins can bind phagocytic
receptors like carcinoembryonic antigen-related cell adhesion molecules (CEACAM). This strain makes an LOS that can incorporate two sialic acid residues. Wild-type or lst mutant Gc was grown with or without addition of CMP-NANA. In the absence of complement, I found sialylated Opa+ Gc decreased the oxidative burst and granule exocytosis from neutrophils. In addition, sialylated Opa+ Gc survived better than nonsialylated or Δlst Gc in the presence of neutrophils within the first hour of infection. However, I unexpectedly found that Gc sialylation did not significantly affect association with or internalization of Gc by neutrophils. LOS sialylation also had no discernable effect on the ability of Opa+ Gc to bind CEACAM3, the activating phagocytic receptor on neutrophils, either as a recombinant protein or a clonal cell line.
Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Siglec-5 and Siglec-9 on neutrophils have inhibitory motifs and have been shown to dampen cellular activity; Siglec-14 is also present in neutrophils but instead is associated with activation. By blocking neutrophil Siglecs with antibodies that bind to their extracellular domains, the ability of sialylated Opa+ Gc to suppress oxidative burst and resist neutrophil killing was eliminated. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea.
One of the challenges of studying sialylation in Gc has been the inability to directly detect sialic acid incorporation on the surface of individual bacteria. To overcome this challenge, we developed two new flow cytometric assays. We were able to expound upon the standard technique of antibody occlusion of a nonsialylated epitope in addition to a novel method to directly tag the sialic acid incorporated by the Gc for labeling using copper-catalyzed azide-alkyne cycloaddition. These methods will aid in the advancement of the field to further our understanding of LOS sialylation in Gc, and potentially other sialylated microbes.

PHD (Doctor of Philosophy)
Neisseria gonorrhoeae, gonorrhea, lipooligosaccharide, neutrophils, sialylation
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