Resistance ofNeisseria gonorrhoeaeto non-oxidative killing by adherent human polymorphonuclear leucocytes

Abstract

SummarySymptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40-70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase-and RecA-deficient Gc, which are more sensitive to ROS in vitro, had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

“…The protocols outlined were used to examine survival of N. gonorrhoeae after exposure to primary human neutrophils 5,26 . Neutrophils were infected with N. gonorrhoeae and processed with protocol 1, using the green-fluorescent viability dye SYTO9 and the red-fluorescent propidium iodide ( Figure 4A).…”

“…Additionally, it can take many hours of electron microscope time to acquire enough micrographs to analyze, while many low-power fields can be acquired from cells exposed to fluorescence-based viability dyes, even in the short time afforded for image acquisition. While the provided protocols were optimized with N. gonorrhoeae and primary human neutrophils, they can be adapted to assess the viability of many species of gram-negative and gram-positive bacteria in a variety of cell types, including all phagocytic and nonphagocytic eukaryotic cells 5,[15][16][17][18][19][20][21][22][23][24]26 . Bacterial viability dyes provide a powerful experimental tool to examine mechanisms of bacterial pathogenesis in eukaryotic cells and the ability of eukaryotic cells to control bacterial infection.…”

“…Colonization triggers the abundant recruitment of neutrophils at mucosal sites. Neutrophils are professional phagocytes that possess a variety of antimicrobial processes to kill microorganisms; however, N. gonorrhoeae is capable of surviving in the presence of neutrophils [2][3][4][5] . Understanding how bacterial pathogens such as N. gonorrhoeae subvert, suppress, and hijack the immune response to ultimately survive in normally hostile host environments is crucial to the development of new therapies for combating infectious diseases.…”

“…The use of these assays provides key insight into the interactions that result in bacterial killing or survival during infection of host cells. The protocols outlined in this article were used to assess the viability of N. gonorrhoeae that is attached to and inside primary human neutrophils, including in different populations of neutrophil phagosomes 5,13,14 . However, these protocols can be applied to assess viability of gram-positive and gram-negative bacteria in professional phagocytes, nonprofessional phagocytes, and protozoa [15][16][17][18][19][20][21][22][23][24] .…”

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

“…We previously showed that some N. gonorrhoeae survive inside primary human neutrophils by residing in immature phagosomes [13]. We have also reported that most extracellular N. gonorrhoeae survive exposure to neutrophils [14], but it was unclear whether this was attributable to any specific mechanism.…”