Survival of Neisseria gonorrhoeae after primary human neutrophil challenge - PROJECT SUMMARY/ABSTRACT The bacterium Neisseria gonorrhoeae (Gc) causes the sexually transmitted infection gonorrhea. With over 82 million cases of gonorrhea estimated worldwide and growing resistance to all clinically recommended antibiotics, the CDC and WHO have labeled Gc an urgent threat “superbug.” The consequences of repeated, untreated, or untreatable Gc infections include infertility, chronic pelvic pain, and ectopic pregnancy. The clinical hallmark of acute gonorrhea is the recruitment of neutrophils to sites of infection. Despite neutrophils’ robust phagocytic and antimicrobial activities, infectious Gc are recovered from neutrophil-rich gonorrheal secretions. Our overarching hypothesis is that Gc possesses mechanisms to evade and subvert the antimicrobial functions of neutrophils, ensuring its persistence in its obligate human host. To test this hypothesis, we model human mucosal infection ex vivo using primary human neutrophils that are adherent and treated with the neutrophil chemoattractant interleukin-8, and we analyze neutrophil-rich specimens from individuals with gonorrhea. In the previous renewal period, we examined how different routes of phagocytosis affect survival of Gc from neutrophils, particularly for bacterial variants in the opacity-associated (Opa) proteins that promote nonopsonic interaction of Gc with neutrophils. We measured a nonstandard distribution of Gc associated with neutrophils ex vivo and in human gonorrheal specimens, where some neutrophils contain numerous Gc and others none. During these studies, we discovered that Gc co-opts C4b-binding protein (C4BP) and sialic acid addition to its lipooligosaccharide to enhance the survival of Opa+ bacteria from neutrophils. C4BP and sialic acid are well-known for conferring resistance of Gc to complement, but our results are complement-independent, instead thwarting CEACAM3- mediated interactions with Opa+ Gc. Along with new ways to modulate Opa expression and abundance, we now have the tools to dissect how Opa+ Gc thwarts CEACAM3-dependent neutrophil activation. In Aim 1 of this renewal application, we will define how C4BP binding to Gc blocks CEACAM3-dependent phagocytosis of Opa+ Gc. In Aim 2, we will delineate how sialylated Gc exploits interaction with sialic acid-binding immunoglobulin-like lectins (Siglecs) to inhibit neutrophil activation. Aim 3 will define heterogeneity in mature human neutrophils’ functions and gene expression profiles, to test the hypotheses that a subset of neutrophils infected with Gc support bacterial survival, with the infecting Gc for each subset correspondingly varying their gene expression profiles. This Aim will integrate multispectral flow cytometric profiling of primary neutrophils, dual RNAseq on infected neutrophils that are flow-sorted and stratified by bacterial load, and neutrophil single-cell RNAseq with targeted functional analysis of bacterial and human gene products. Our multidisciplinary approach combines expertise in Gc pathogenesis, mass spectrometry, complement inhibition, bioinformatics, and clinical infectious diseases. Through these ongoing studies on how Gc subverts neutrophil attack, new host or bacterial targets for therapeutics can be identified, to combat the global public health burden of drug-resistant gonorrhea.
