Interactions of Neisseria gonorrhoeae with Human Neutrophils: Effects of Serum and Gonococcal Opacity on Phagocyte Killing and Chemiluminescence

Abstract

Serum-sensitive strains of Neisseria gonorrhoeae were incubated with suspensions of normal or chronic granulomatous disease human neutrophils in the absence or presence of fresh or heat-inactivated human serum; phagocytosis, gonococcal viability, and chemiluminescence were measured. Nonpiliated opaque or transparent gonococci (colony types 3 and 4, respectively) were used for phagocytic bactericidal assays. In the presence of 2.0% fresh human serum, normal neutrophils killed >90% of types 3 and 4 gonococci by 135 min. Serum alone at this concentration was not bactericidal. In the absence of serum, type 4 gonococci were not killed, whereas type 3 gonococci were killed to the same degree as in the presence of serum. Interestingly, heat-inactivated normal serum slightly inhibited phagocytic killing of type 3 gonococci. Results almost identical to those above were obtained when 5% fresh human serum deficient in complement component 7 was substituted for 2% normal autologous serum. This indicated that the later components of complement were not involved in the observed results. To investigate the mechanisms responsible for the intracellular killing of the gonococci, we used neutrophils from patients with chronic granulomatous disease. These neutrophils are deficient in an activable NADPH oxidase and do not produce bactericidal oxygen products upon phagocytic stimulation. Neutrophils from two unrelated boys with chronic granulomatous disease killed type 3 and 4 gonococci to the same degree as did normal neutrophils. As with normal neutrophils, serum was needed for killing type 4 organisms. As expected, neutrophils from these patients showed absolutely no increased chemiluminescence in the presence of type 3 or 4 gonococci, with or without serum. The effects of serum on gonococcus-induced chemiluminescence by normal neutrophils was also investigated. For these studies, in addition to type 3 and 4 gonococci, we also used transparent colony types of lightly (type 1) and heavily (type 2) piliated organisms. Chemiluminescence induced by type 1, 2, or 3 gonococci (i.e., gonococci possessing either pili or opacity-associated proteins, but not both) was augmented only slightly by serum and then only at low ratios of gonococci to neutrophils. On the other hand, chemiluminescence induced by type 4 gonococci (i.e., gonococci possessing neither pili nor opacity-associated proteins) was substantially increased in the presence of serum. Stimulation of chemiluminescence by type 1, 2, 3, or 4 gonococci was dose dependent in the absence or presence of serum. Heat-killed type 3 gonococci induced chemiluminescence to the same degree as did viable organisms. Since the gonococci used in this research was strongly catalase positive, as are gonococci in general, and since it was killed by chronic granulomatous disease neutrophils, the results indicate that gonococci can be effectively killed within neutrophils, i.e., within phagolysosomes, by nonoxidative bactericidal mechanisms. Whereas type 3 gonococci were phagocytized and killed by neutr...

Neisseria gonorrhoeaesuppresses the oxidative burst of human polymorphonuclear leukocytes

“…Alternatively, they may indicate that PMNs primarily direct non-oxidative antimicrobial mechanisms against Gc. This possibility is supported by reports from the Rest labora-tory showing that PMNs maintained under anoxic conditions or lacking NADPH oxidase activity retain some ability to kill Opa + Gc (Rest et al, 1982;Frangipane and Rest, 1992), as well as the recent observation that Gc mutated in genes encoding various antioxidants are not compromised for survival after PMN challenge (Seib et al, 2005). We are currently re-examining which antimicrobial mechanisms PMNs direct against Gc during infection.…”

“…type IV pili) that could potentially facilitate Opa-independent interactions with PMNs (Rest et al, 1982;Virji and Heckels, 1986;Fischer and Rest, 1988;Elkins and Rest, 1990). In re-examining the data in these reports, Opa -Gc do appear to trigger some degree of ROS production from PMNs, albeit less than their Opa + counterparts (Rest et al, 1982;Virji and Heckels, 1986). Given these discrepancies in the literature, it is apparent that key questions remain regarding PMN responsiveness to Gc infection.…”

“…The weak ROS response of PMNs to liquid-grown OpaB + Gc led us to consider whether bacterial growth conditions influenced the ability of PMNs to mount an oxidative burst. Previous work examining Opa + Gc interactions with PMNs typically used bacteria collected from agarcontaining medium or grown in liquid culture for short times (< 3 h) prior to infection (Rest et al, 1982 A. Minor oxidative burst in PMNs infected with live, exponentially growing OpaB + FA1090 Gc.…”

Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili

“…Second, a selection for P+ Gc may occur during the inflammatory response of gonorrhoeal disease. Piliated Gc may be enriched in secretions and urine during symptomatic disease because they are more resistant to the bactericidal mechanisms of leucocytes (Rest et al, 1982). Third, P+ Gc may survive better in nutrient-limiting conditions in primary epithelial cells or the urogenital lumen (but not T84 cells), because the pilus can mediate acquisition of haem-containing compounds and potentially other nutrients (Chen et al, 2004).…”

Immunobiology of purified recombinant outer membrane porin protein I of Neisseria gonorrhoeae

“…The ability of anti-rPor antisera to induce phagocytic kiiiing of GC by PMNs was measured using assays described by Rest et al (1982), Wetzler et at. (1988) and Leifh ef al.…”