Phosphoethanolamine decoration of Neisseria gonorrhoeae lipid A plays a dual immunostimulatory and protective role during experimental genital tract infection

Abstract

The induction of an intense inflammatory response by Neisseria gonorrhoeae and the persistence of this pathogen in the presence of innate effectors is a fascinating aspect of gonorrhea. Phosphoethanolamine (PEA) decoration of lipid A increases gonococcal resistance to complement-mediated bacteriolysis and cationic antimicrobial peptides (CAMPs), and recently we reported that wild-type N. gonorrhoeae strain FA1090 has a survival advantage relative to a PEA transferase A (lptA) mutant in the human urethral-challenge and murine lower genital tract infection models. Here we tested the immunostimulatory role of this lipid A modification. Purified lipooligosaccharide (LOS) containing lipid A devoid of the PEA modification and an lptA mutant of strain FA19 induced significantly lower levels of NF-κB in human embryonic kidney Toll-like receptor 4 (TLR4) cells and murine embryonic fibroblasts than wild-type LOS of the parent strain. Moreover, vaginal proinflammatory cytokines and chemokines were not elevated in female mice infected with the isogenic lptA mutant, in contrast to mice infected with the wild-type and complemented lptA mutant bacteria. We also demonstrated that lptA mutant bacteria were more susceptible to human and murine cathelicidins due to increased binding by these peptides and that the differential induction of NF-κB by wild-type and unmodified lipid A was more pronounced in the presence of CAMPs. This work demonstrates that PEA decoration of lipid A plays both protective and immunostimulatory roles and that host-derived CAMPs may further reduce the capacity of PEA-deficient lipid A to interact with TLR4 during infection.

FIG 1

Gonococcal LOS with PEA-decorated lipid A but not unmodified lipid A induces NF-κB in HEK (TLR4) and MEFs. Human and murine cell lines carrying an NF-κB SEAP reporter gene were incubated with increasing doses of purified LOS from wild-type (shaded bars) or lptA mutant (open bars) bacteria. SEAP activity was measured after 20 h of incubation. (A) HEK cells expressing the human TLR4 receptor complex; (B) MEFs from C3H/HeN mice. SEAP activity was plotted as an OD₆₅₀ value. Null2 HEK cells that are not transfected with the human TLR4 receptor complex induced minimal SEAP activity in response to purified LOS that was comparable to the level of SEAP activity detected in wells to which no LOS was added (data not shown). Asterisks indicate differences for which the level of significance was <0.05.

FIG 2

Whole wild-type and lptA mutant gonococci induce similar levels of NF-κB in MEFs, but the lptA mutant is preferentially killed by supernatants from MEFs incubated with wild-type gonococci. Whole bacteria were incubated with MEFs carrying an NF-κB SEAP reporter gene, and induction of NF-κB expression, bacterial viability, and cell death were measured after 24 h of incubation. (A) SEAP activity; (B) average numbers of bacteria recovered from triplicate wells; (C) LDH activity to measure MEF viability. The three strains grew equally well in the tissue culture medium, and minimal cell death occurred during the assay (cell death was comparable to that in wells with MEFs alone).

FIG 3

Wild-type and lptA mutant gonococci colonize mice equally well during noncompetitive infections. Groups of BALB/c mice were inoculated with 10⁵ CFU of wild-type, lptA mutant, or C′lptA mutant bacteria, and the number of bacteria recovered from vaginal swabs was determined by quantitative culture (n = 11 to 16 mice per strain).

FIG 4

Gonococci devoid of the PEA lipid A moiety do not induce a proinflammatory response. Vaginal cytokines and chemokines were measured on day 5 postinoculation in the noncompetitive experiments whose results are shown in Fig. 3. Horizontal bars indicate the geometric mean. Kw*, Kruskal-Wallis test. P < 0.05, Dunn's multiple comparison.

FIG 5

The lipid A PEA modification provides a competitive advantage during experimental genital tract infection. Female BALB/c mice were inoculated vaginally with similar numbers of bacteria of wild-type FA19Sm^R and the lptA mutant (A) or wild-type FA19Sm^R and the C′lptA mutant (B), and the relative recovery of each strain was determined over time. The competitive index (CI) for each mouse at each time point is shown, with the geometric mean indicated by a horizontal bar. A CI of 1.0 indicates equal levels of fitness, a CI of <1.0 indicates reduced fitness of the mutants, and a CI of >1.0 indicates greater fitness of the mutants. The limit of detection (4 CFU) was used for the number of mutant or wild-type gonococci when these strains were not recovered. Open diamonds correspond to mice in which only wild-type bacteria (A) or C′lptA mutant bacteria (B) were recovered. The open circle in panel A corresponds to a time point from which only mutant bacteria were recovered from one mouse. The decrease in the number of data points over time is due to some mice clearing infection or being culture negative at that time point. Results are the combined data from three different experiments.

FIG 6

PEA-decorated lipid A protects N. gonorrhoeae from cathelicidins. A semiquantitative assay was used to assess bacterial susceptibility to human and murine cathelicidins. A scrambled LL-37 peptide was used as a negative control. hLL-37, human LL-37.

FIG 7

Cathelicidins preferentially bind to gonococci with lipid A devoid of the 4′ PEA moiety. Flow cytometry was used to measure surface binding of CRAMP-38 and LL-37 wild-type, lptA, and C′lptA mutant bacteria. The open and shaded areas represent the presence and absence of CAMPS, respectively. The assay was performed three times, and the results were similar.

FIG 8

Preincubation with CAMPs differentially neutralizes the inflammatory potential of unmodified LOS. LOS from wild-type (filled bars) or lptA mutant (open bars) gonococci was incubated with PMB (10 μg/ml) or 10 μg of CRAMP-38, human LL-37, or lysozyme and then tested for its capacity to induce NF-κB expression in MEFs or bind to the LAL reagent. (A) SEAP activity produced by MEFs; (B) LAL activity. Results using 1 μg/ml and 100 μg/ml of PMB were similar (data not shown). Single, double, and triple asterisks correspond to P values that are ≤0.05, ≤0.01, and ≤0.001, respectively.