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, 73 (4), 1971-7

Interactions of Neisseria Gonorrhoeae With Adherent Polymorphonuclear Leukocytes

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Interactions of Neisseria Gonorrhoeae With Adherent Polymorphonuclear Leukocytes

Mark P Simons et al. Infect Immun.

Abstract

Neisseria gonorrhoeae causes severe exudative urethritis. The exudates from infected individuals contain large numbers of polymorphonuclear leukocytes (PMN) with ingested gonococci. The fate of N. gonorrhoeae within PMN has been a topic of debate for years. In this study, we examined the interactions of N. gonorrhoeae with PMN adherent to surfaces as a system that better models events during clinical disease. Using chemiluminescence to measure reactive oxygen species (ROS), we found that N. gonorrhoeae stimulated PMN to produce a respiratory burst. Different kinetics were seen when PMN were stimulated with opsonized zymosan particles. In addition, ROS were produced predominantly inside the PMN in response to gonococci. Laser scanning confocal microscopy and transmission electron microscopy showed that N. gonorrhoeae rapidly associated with PMN under these experimental conditions and was internalized. Some gonococci were cleared in the first 30 to 60 min after phagocytosis, but a majority of the population persisted for 6 h after phagocytosis. Quantification of viable organisms showed that a significant portion of the population resisted killing. The viability of this subpopulation remained unchanged for 2 h after phagocytosis. A significant increase of viable gonococci from 1 to 6 h was also observed, suggesting intracellular replication. Four different N. gonorrhoeae strains demonstrated the same capacity to resist PMN-mediated killing, whereas Escherichia coli was rapidly killed by PMN under the same conditions. Taken together, these findings suggest that a subpopulation of N. gonorrhoeae resists killing and replicates within PMN phagosomes in spite of NADPH oxidase activation.

Figures

FIG. 1.
FIG. 1.
Chemiluminescence. Adherent PMN were stimulated in the presence of lucigenin (A), luminol (B), or isoluminol (C) with 1:1 OPZ (▪) or the following dose ranges of N. gonorrhoeae strain 1291: 1:1 (▵), 10:1 (□), 20:1 (×), 50:1 (♦), 100:1 (+). Unstimulated, resting cells (⋄) were used as a negative control. For isoluminol chemiluminescence, 5:1 OPZ (▴) was used to stimulate a larger response. Chemiluminescence readings were taken every 2 min for 1 h. Data are the result of a single representative experiment, with each value presented as the mean of three replicates.
FIG. 2.
FIG. 2.
N. gonorrhoeae associated with PMN. (A and B) PMN were challenged with N. gonorrhoeae strain 1291gfp at an MOI of 10:1 for 15 min and examined by LSCM (A) and SEM (B). The LSCM image was viewed at 630× magnification, with GFP-containing bacteria seen using the FITC fluorescence channel and PMN viewed by DIC. SEM images were viewed at 1,000× magnification. (C) PMN were challenged with N. gonorrhoeae strain 1291gfp at an MOI of 10:1 for the indicated times, washed, fixed with 2% paraformaldehyde, and examined by LSCM. Cell-associated gonococci were counted, and PMN were grouped according to the numbers of associated gonococci: 0 gonococci/PMN (blue), 1 to 9 gonococci/PMN (white), or ≥10 gonococci/PMN (black). Values are the percentages of total PMN counted at each time point.
FIG. 3.
FIG. 3.
N. gonorrhoeae was internalized by PMN. PMN were challenged with N. gonorrhoeae strain 1291 at an MOI of 10:1 and processed for TEM. PMN were challenged for 1 h (A; 8,000×), 2 h (B; 12,000×), 4 h (C; 8,000×), or 6 h (D; 7,000×). Gonococci appear within PMN phagosomes.
FIG. 4.
FIG. 4.
N. gonorrhoeae survived killing and replicated within PMN. (A) PMN were challenged with N. gonorrhoeae strain 1291 (▴) or E. coli (▪) for 2 h at an MOI of 1:1. PMN were lysed at 30-min intervals, serial diluted, and plated for CFU counts. Results are shown as the percentage of viable bacteria present at T0. Data are the result of a single representative experiment, and values are presented as the mean of three replicates with the standard error of the mean. (B) PMN were challenged with N. gonorrhoeae 1291 at an MOI of 1:1 for 1 and 6 h and lysed to quantify CFU. Results are shown as the percentage of viable bacteria at T0. Data are the result of a single representative experiment, with values presented as the mean of three replicates and the standard error of the mean. Differences were statistically significant (P = 0.0196).
FIG. 5.
FIG. 5.
N. gonorrhoeae strain comparison. PMN were challenged with N. gonorrhoeae strains 1291 (A), FA1090 (B), F62 (C), or PID2 (D) for 1 and 2 h at an MOI of 1:1. PMN were lysed to quantify CFU. Results are shown as the percentage of viable bacteria at T0. Each data point is the mean of three replicates performed on individual days.

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