Constitutively Opa-Expressing and Opa-Deficient Neisseria gonorrhoeae Strains Differentially Stimulate and Survive Exposure to Human Neutrophils

Abstract

The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in frame, termed Opaless. Opaless Gc remained uniformly Opa negative (Opa ؊ ), whereas cultures of predominantly Opa ؊ parental Gc and an intermediate lacking the "translucent" subset of opa genes (⌬opaBEGK) stochastically gave rise to Opa-positive (Opa ؉ ) bacterial colonies. Loss of Opa expression did not affect Gc growth. Opaless Gc survived exposure to primary human PMNs and suppressed the PMN oxidative burst akin to parental, Opa ؊ bacteria. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. When a non-phase-variable, in-frame allele of FA1090 opaD was reintroduced into Opaless Gc, the bacteria induced the PMN oxidative burst, and OpaD ؉ Gc survived less well after exposure to PMNs compared to Opa ؊ bacteria. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology.

A Thermonuclease ofNeisseria gonorrhoeaeEnhances Bacterial Escape From Killing by Neutrophil Extracellular Traps

“…NET integrity was comparable between parent and Δnuc N. gonorrhoeae at times up to 1 hour, after which it decreased with parent N. gonorrhoeae but increased in Δnuc-exposed cells (Figure 2). Consistent with previous findings for this opa-deficient background [18], neither Nuc-containing nor Δnuc N. gonorrhoeae induced production of reactive oxygen species by neutrophils (Supplementary Figure 1C), indicating that effects on NET integrity were not due to differences in the neutrophil oxidative burst. Together, these results show N. gonorrhoeae induces rapid formation of NETs by human neutrophils and that NET integrity decreases over time with Nuc-containing N. gonorrhoeae.…”

“…Piliated, opacity protein-deficient N. gonorrhoeae (strain FA1090) was used for most experiments [18]. An insertiondeletion mutation with a kanamycin resistance cassette was generated in nuc (NGO0969), using plasmid pCTS#43 [17].…”

Selection for a CEACAM Receptor-Specific Binding Phenotype during Neisseria gonorrhoeae Infection of the Human Genital Tract

“…Characterization of individual CEACAM function in neutrophilic cell lines showed that Opa-mediated neutrophil activation is mediated by CEACAM3 signaling, while CEACAM1 and CEACAM6 allowed bacterial internalization without triggering production of reactive oxygen species (ROS) or release of toxic granule proteins (44). However, all of the studies focusing on neutrophil responses to Opa-CEACAM binding have been done exclusively with the commonly studied N. gonorrhoeae strains MS11 (44,45,47,61,62) and FA1090 (63,64).…”

Neisseria gonorrhoeae Evades Calprotectin-Mediated Nutritional Immunity and Survives Neutrophil Extracellular Traps by Production of TdfH

“…Gonococcal opacity (Opa) proteins, which are subject to phase variation, have been shown to differentially affect gonococcal survival in association with neutrophils (16). Therefore, all neutrophil experiments were conducted in the FA1090 Opa-deficient (Opaless) background (43). Primary human neutrophils seeded onto chambered cover glasses and induced to form NETs with phorbol myristate acetate (PMA) (53) were stained with antibodies against CP and gonococci.…”