Alpha-2,3-sialyltransferase enhances Neisseria gonorrhoeae survival during experimental murine genital tract infection

Abstract

The addition of host-derived sialic acid to Neisseria gonorrhoeae lipooligosaccharide is hypothesized to be an important mechanism by which gonococci evade host innate defenses. This hypothesis is based primarily on in vitro assays of complement-mediated and phagocytic killing. Here we report that a nonpolar alpha-2,3-sialyltransferase (lst) mutant of N. gonorrhoeae was significantly attenuated in its capacity to colonize the lower genital tract of 17-beta estradiol-treated female BALB/c mice during competitive infection with the wild-type strain. Genetic complementation of the lst mutation restored recovery of the mutant to wild-type levels. Studies with B10.D2-HC(o)H2(d)H(2)-T18c/OSN (C5-deficient) mice showed that attenuation of the lst mutant was not due to increased sensitivity to complement-mediated bacteriolysis, a result that is consistent with recently reported host restrictions in the complement cascade. However, Lst-deficient gonococci were killed more rapidly than sialylated wild-type gonococci following intraperitoneal injection into normal mice, which is consistent with sialylation conferring protection against killing by polymorphonuclear leukocytes (PMNs). As reported for human PMNs, sialylated gonococci were more resistant to killing by murine PMNs, and sialylation led to reduced association with and induction of a weaker respiratory burst in PMNs from estradiol-treated mice. In summary, these studies suggest sialylation confers a survival advantage to N. gonorrhoeae in mice by increasing resistance to PMN killing. This report is the first direct demonstration that alpha-2,3-sialyltransferase contributes to N. gonorrhoeae pathogenesis in an in vivo model. This study also validates the use of experimental murine infection to study certain aspects of gonococcal pathogenesis.

FIG. 1.

Serum resistance of in vivo- and in vitro-grown wild-type N. gonorrhoeae. The number of viable gonococci recovered following incubation in increasing concentrations of NHS is shown for wild-type MS11 gonococci tested directly in vaginal swab suspensions or following in vitro subculture in the presence or absence of CMP-NANA. Of all conditions tested, only gonococci within vaginal swab suspensions and/or isolated from in vitro culture in the presence of CMP-NANA exhibited high levels of serum resistance. NANase was used to confirm the basis of the serum resistance.

FIG. 2.

Recovery of the lst mutant (GP300) or complemented mutant (GP322) relative to the wild-type strain. Results are expressed as competitive indices (CI) from individual mice at each time point. The CI was calculated as described in Materials and Methods. The ratios of mutant to wild-type gonococci in the inoculum for all experiments were 0.47 to 0.50. Horizontal bars represent the geometric mean; a CI of <1.0 indicates a decrease in the ratio of mutant to wild-type gonococci with respect to that of the inoculum. Open symbols represent mice from which at least 100 wild-type CFU, but no GP300 or GP322 CFU, were recovered; the limit of detection (0.04 CFU per 100 μl of swab suspension) was used as the number of mutant CFU recovered in these cases.

FIG. 3.

Susceptibility of wild-type (MS11), Lst-deficient (GP300), or complemented lst mutant (GP322) to opsonophagocytic killing by murine PMNs. Gonococci cultured in the presence or absence of CMP-NANA or with CMP-NANA followed by NANase treatment were preincubated with NMS (opsonized) or HI-NMS (control) and then incubated with murine PMNs. GP322 was cultured without IPTG for these experiments. Results are expressed as the mean number of CFU recovered after 90 min of incubation; standard error bars are based on samples cultured in quadruplicate. *, P < 0.05 compared to HI-NMS control; **, P < 0.005 compared to HI-NMS control.

FIG. 4.

Association of sialylated or nonsialylated gonococci with murine PMNs. Results were quantitated by examining 100 PMNs for each of three coverslips stained with acridine orange and determining the average number of PMNs associated with gonococci and the number of gonococci per PMN. A. Average number of PMNs associated with wild-type MS11 gonococci preincubated with NMS (opsonized) or HI-NMS. Standard error bars are shown. Significantly more PMNs were associated with opsonized gonococci (P < 0.05; unpaired t test) regardless of whether MS11 was cultured with or without CMP-NANA. B. Average number of PMNs associated with 0, 1 to 2, 3 to 10, or >10 MS11 or GP322 gonococci cultured in the presence or absence of CMP-NANA or GP300 gonococci cultured with CMP-NANA. All gonococci were preincubated in NMS. The average total number of PMNs associated with MS11 or GP322 gonococci cultured in CMP-NANA (and without IPTG) was statistically different from that detected for nonsialylated MS11, GP322, or GP300 (P < 0.05; unpaired t test).

FIG. 5.

Oxidative response of mouse PMNs to sialylated and nonsialylated gonococci. Isoluminol- or luminol-enhanced CL assays were utilized to measure the total (top panel), intracellular (middle panel), and extracellular (bottom panel) release of ROS by PMNs from estradiol-treated mice following exposure to wild-type MS11, lst mutant GP300, and complemented strain GP322, as described in Materials and Methods. Bacteria were cultured in CMP-NANA; IPTG was not used to maximally induce the complementing lst gene in GP322.

FIG. 6.

Recovery of the lst mutant (GP300) from the C5-deficient and C5-sufficient mice relative to the wild-type strain. C5⁻ results are expressed as competitive indices (CI) for individual C5⁻ (n = 4) and C5⁺ (n = 6) mice as described in the Fig. 2 legend. Horizontal bars represent the mean CI. The ratio of mutant to wild-type gonococci in the inoculum was 0.5. Cultures from which no GP300 gonococci were recovered are represented by open symbols.

FIG. 7.

Survival of sialylated and nonsialylated gonococci following i.p. injection of estradiol-treated mice. Wild-type MS11 and lst mutant GP300 gonococci cultured in CMP-NANA were used in these assays. The mutant was significantly killed compared to results for the wild-type strain at 4 and 5 h (P < 0.0001 and P = 0.02, respectively).