A Novel Sialylation Site on Neisseria gonorrhoeae Lipooligosaccharide Links Heptose II Lactose Expression with Pathogenicity

Abstract

Sialylation of lacto--neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for monoclonal antibody (MAb) 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho--acetylneuraminic acid (CMP-Neu5Ac)-containing medium enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOSs of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and an MS11 mutant with lactose only from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by α2-3-specific neuraminidase suggested an α2-6 linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. Strain 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with MAb 2C7; MAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. MAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose Our findings reinforce the candidacy of the 2C7 epitope as a vaccine antigen and MAb 2C7 as an immunotherapeutic antibody.

Structure of the Recombinant Neisseria gonorrhoeae Adhesin Complex Protein (rNg-ACP) and Generation of Murine Antibodies with Bactericidal Activity against Gonococci

“…It has been well documented that LOS sialylation prevents complement-dependent killing by immune sera generated in response to different OM antigens ( 33 , 34 , 46 ). More recently, Ram and colleagues demonstrated how increasing amounts of CMP-NANA sialylation reduced the bactericidal activity of the 2C7 monoclonal antibody ( 47 ). Concentrations of >3 µg/ml of CMP-NANA led to >50% survival of gonococci in response to the effects of the bactericidal antibody.…”

Role of Gonococcal Neisserial Surface Protein A (NspA) in Serum Resistance and Comparison of Its Factor H Binding Properties with Those of Its Meningococcal Counterpart

“…N. gonorrhoeae strains often express multiple LOS structures simultaneously (67,68). A recent survey of 75 minimally passaged N. gonorrhoeae isolates obtained from men who attended a sexually transmitted diseases clinic in Nanjing, China, revealed that 100% of isolates expressed LNnT but also simultaneously expressed LOS with only lactose extensions from HepI, evidenced by reactivity with mAb 2-1-L8 (69)(70)(71). The ability to retain FH binding even in the absence of LNnT sialyation, in this instance by binding of FH SCR domains 6 and 7 by NspA, will enable complement evasion.…”

Bypassing Phase Variation of Lipooligosaccharide (LOS): Using Heptose 1 Glycan Mutants To Establish Widespread Efficacy of Gonococcal Anti-LOS Monoclonal Antibody 2C7

“…As a result, such attenuated escape mutants would be cleared by host immune defenses. We previously identified a murine monoclonal antibody (MAb), 2C7 (2C7-murine), that reacted with 95% or 100% of N. gonorrhoeae recovered directly from cervical secretions (7) or minimally passaged clinical isolates (10), respectively, and shortened the duration and reduced the burden of infection in mice (8). The minimal structure required for MAb 2C7 binding (i.e., the 2C7 epitope) requires lactose extensions simultaneously from the heptose I (HepI) and HepII core glycans (see Fig.…”

“…S1). Expression of HepII lactose facilitates colonization of the mouse vagina by gonococci; lgtG deletion mutants are attenuated in mice (8,10).…”

“…We recently discovered that the lactose extension from HepII could be replaced with N-acetylneuraminic acid (Neu5Ac), which inhibited complement C3 fragment deposition (10) and engaged Siglec receptors (11), which may downregulate the host inflammatory response to infection. As stated above, resistance to MAb 2C7 because of lgtG being phase-varied off would result in considerable loss of bacterial fitness.…”