Neisseria gonorrhoeae (Gc) is an obligate human bacterial pathogen and the cause of gonorrhea. Gc is a major public health concern because numbers of cases remain high, it has gained resistance to multiple antibiotics, no vaccine exists, and infection is associated with debilitating, lifelong negative outcomes such as infertility and ectopic pregnancy. Mucosal colonization by Gc drives the recruitment of polymorphonuclear leukocytes (PMNs or neutrophils) to the site of infection and the production of a characteristic purulent exudate. Despite PMNs? robust phagocytic and antimicrobial activities, infectious Gc can be cultured from PMN-rich exudates, implying Gc possesses mechanisms to evade killing by PMNs. The failure of PMNs to clear Gc not only allows the infection to persist, but also sustains the PMN inflammatory response, causing the tissue damage that underlies infertility and other sequelae. To identify the mechanisms used by Gc to resist killing by PMNs, we developed an infection model using adherent, chemokine-treated primary human PMNs to approximate the physiological state of PMNs recruited to mucosal surfaces. We used this model to uncover two major ways in which Gc survives after exposure to PMNs. First, Gc expresses gene products that confer resistance to PMN antimicrobial compounds, such as proteases and antimicrobial peptides. Second, Gc varies expression of surface structures to modulate the route of phagocytosis by PMNs and consequent exposure to these compounds. Specifically, Gc that have phase-varied off expression of opacity-associated (Opa) proteins is internalized into immature phagosomes in which it survives and suppresses the PMN oxidative burst, while Gc expressing an Opa protein that interacts with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) 1 and 3 is phagocytosed into a mature, degradative phagolysosome and induces a potent oxidative burst. In this renewal proposal, we will continue to dissect the mechanisms that underlie Gc survival after PMN challenge.
Aim 1 will define how Opa-negative Gc is phagocytosed into PMNs without eliciting the oxidative burst or phagolyososome formation and will evaluate the survival profile of Gc expressing an Opa protein that does not bind CEACAM3. We will evaluate the long-term consequences of these receptor- mediated interactions on the viability of both Gc and PMNs.
Aim 2 will build upon results from a Tn-seq experiment conducted in the previous funding period to examine how bacterial efflux pumps and lipooligosaccharide variants contribute to Gc survival from PMNs. All experiments will be conducted with the adherent, interleukin-8 treated PMN infection model alongside analysis of freshly collected human gonorrheal exudates. Together, the results from this research will reveal the diverse approaches used by Gc to survive from PMNs, which ensures the continued persistence of gonorrhea in the human population even when facing a neutrophilic inflammatory response. This research can point to targets in Gc or PMNs that can be exploited for new therapies to treat drug-resistant gonorrhea while limiting the host damage associated with PMN influx.

Public Health Relevance

Gonorrhea affects over 106 million people worldwide every year, and it is an ?urgent? health threat because the bacteria causing gonorrhea are now resistant to most or all recommended antibiotics. Untreated gonorrhea leads to pelvic inflammatory disease and ectopic pregnancy in women, blindness in newborns, and infertility in men and women. Our research explores how gonorrhea bacteria survive after encountering neutrophils during the initial immune response, which may reveal new targets (in human cells or in the bacteria) for developing effective therapeutics to treat multidrug-resistant gonorrhea.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Host Interactions with Bacterial Pathogens Study Section (HIBP)
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Vincent, Leah Rebecca
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University of Virginia
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Palmer, Allison; Criss, Alison K (2018) Gonococcal Defenses against Antimicrobial Activities of Neutrophils. Trends Microbiol 26:1022-1034
Ragland, Stephanie A; Humbert, Mar?a V; Christodoulides, Myron et al. (2018) Neisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme. PLoS Pathog 14:e1007080
Stevens, Jacqueline S; Gray, Mary C; Morisseau, Christophe et al. (2018) Endocervical and Neutrophil Lipoxygenases Coordinate Neutrophil Transepithelial Migration to Neisseria gonorrhoeae. J Infect Dis 218:1663-1674
Handing, Jonathan W; Ragland, Stephanie A; Bharathan, Urmila V et al. (2018) The MtrCDE Efflux Pump Contributes to Survival of Neisseria gonorrhoeae From Human Neutrophils and Their Antimicrobial Components. Front Microbiol 9:2688
Stevens, Jacqueline S; Criss, Alison K (2018) Pathogenesis of Neisseria gonorrhoeae in the female reproductive tract: neutrophilic host response, sustained infection, and clinical sequelae. Curr Opin Hematol 25:13-21
Ragland, Stephanie A; Schaub, Ryan E; Hackett, Kathleen T et al. (2017) Two lytic transglycosylases in Neisseria gonorrhoeae impart resistance to killing by lysozyme and human neutrophils. Cell Microbiol 19:
Smirnov, Asya; Solga, Michael D; Lannigan, Joanne et al. (2017) High-Throughput Particle Uptake Analysis by Imaging Flow Cytometry. Curr Protoc Cytom 80:11.22.1-11.22.17
Ragland, Stephanie A; Criss, Alison K (2017) From bacterial killing to immune modulation: Recent insights into the functions of lysozyme. PLoS Pathog 13:e1006512
Jean, Sophonie; Juneau, Richard A; Criss, Alison K et al. (2016) Neisseria gonorrhoeae Evades Calprotectin-Mediated Nutritional Immunity and Survives Neutrophil Extracellular Traps by Production of TdfH. Infect Immun 84:2982-94
Martin, Jennifer N; Ball, Louise M; Solomon, Tsega L et al. (2016) Neisserial Opa Protein-CEACAM Interactions: Competition for Receptors as a Means of Bacterial Invasion and Pathogenesis. Biochemistry 55:4286-94

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