Sialylation of lipooligosaccharides is dispensable for the virulence of Haemophilus ducreyi in humans

Abstract

Sialylated glycoconjugates on the surfaces of mammalian cells play important roles in intercellular communication and self-recognition. The sialic acid preferentially expressed in human tissues is N-acetylneuraminic acid (Neu5Ac). In a process called molecular mimicry, many bacterial pathogens decorate their cell surface glycolipids with Neu5Ac. Incorporation of Neu5Ac into bacterial glycolipids promotes bacterial interactions with host cell receptors called Siglecs. These interactions affect bacterial adherence, resistance to serum killing and phagocytosis, and innate immune responses. Haemophilus ducreyi, the etiologic agent of chancroid, expresses lipooligosaccharides (LOS) that are highly sialylated. However, an H. ducreyi sialyltransferase (lst) mutant, whose LOS contain reduced levels of Neu5Ac, is fully virulent in human volunteers. Recently, a second sialyltransferase gene (Hd0053) was discovered in H. ducreyi, raising the possibility that Hd0053 compensated for the loss of lst during human infection. CMP-Neu5Ac is the obligate nucleotide sugar donor for all bacterial sialyltransferases; LOS derived from an H. ducreyi CMP-Neu5Ac synthetase (neuA) mutant has no detectable Neu5Ac. Here, we compared an H. ducreyi neuA mutant to its wild-type parent in several models of pathogenesis. In human inoculation experiments, the neuA mutant formed papules and pustules at rates that were no different than those of its parent. When grown in media with and without Neu5Ac supplementation, the neuA mutant and its parent had similar phenotypes in bactericidal, macrophage uptake, and dendritic cell activation assays. Although we cannot preclude a contribution of LOS sialylation to ulcerative disease, these data strongly suggest that sialylation of LOS is dispensable for H. ducreyi pathogenesis in humans.

Fig 1

Silver-stained gel of LOS isolated from 35000HP (lanes 1 and 2) and the neuA mutant (lanes 3 and 4). The bacteria were grown in broth without (lanes 1 and 3) and with (lanes 2 and 4) CMP-Neu5Ac supplementation. Note that under both growth conditions LOS derived from 35000HP contains the a-branch (∗), while LOS derived from the neuA mutant contains only the b-branch (∗∗).

Fig 2

Bactericidal assays. (A) Percent survival of 35000HP, 35000HP-RSM208 (neuA mutant), 35000HP-RSM203 (lst mutant), and FX517 (dsrA mutant) in 50% NHS, calculated as follows: [(geometric mean CFU in active NHS/geometric mean CFU in heat-inactivated NHS) × 100]. P values are as follows: for 35000HP versus 35000HP-RSM208, P = 0.22; for 35000HP versus 35000HP-RSM203, P = 0.25; for 35000HP versus FX517, P = 0.001; for 35000HP-RSM208 versus 35000HP-RSM203, P = 0.057; for 35000HP-RSM208 versus FX517, P = 0.0004; and for 35000HP-RSM203 versus FX517, P = 0.0002. (B) Percent survival of 35000HP, 35000HP-RSM208, and FX517 grown in media supplemented with 1 mM Neu5Ac and FX517 grown without Neu5Ac supplementation in 50% NHS, calculated as described for panel A. P values are as follows: for 35000HP versus 35000HP-RSM208, P = 0.059; for 35000HP versus FX517 grown with Neu5Ac, P = 0.006; for 35000HP versus FX517 grown without Neu5Ac, P = 0.0001; for 35000HP-RSM208 versus FX517 grown with Neu5Ac, P < 0.0001; for 35000HP-RSM208 versus FX517 without Neu5Ac, P < 0.0001; and for FX517 grown with Neu5Ac versus FX517 grown without Neu5Ac, P = 0.16. For both panels, values are means ± SDs from 5 independent experiments.

Fig 3

Percentages of H. ducreyi that were associated with (top) or internalized by (bottom) macrophages. (A) 35000HP and 35000HP-RSM208 (neuA mutant) grown on chocolate agar plates with and without Neu5Ac supplementation were incubated with macrophages at an MOI of 10:1 for 30 min without opsonization, followed by 30 min of treatment with gentamicin. Percent association or internalization was calculated as the ratio of bacteria recovered without or with gentamicin treatment to initial CFU added. The values represent the means ± SDs from assays done with macrophages from 5 (top) or 6 (bottom) donors. There were no significant differences among the groups in either assay. (B) 35000HP and 35000HP-RSM208 were grown on chocolate agar plates with Neu5Ac supplementation and were either not opsonized or opsonized with 100% autologous serum prior to performance of the assays outlined for panel A. The values in both panels represent the means ± SDs from assays done with macrophages from 4 donors. There were no significant differences between the neuA mutant and the parent strain in either assay.

Fig 4

Cytokine production by DC. DC were uninfected or infected with 35000HP and 35000HP-RSM208 (neuA mutant) grown on chocolate agar plates with and without 1 mM Neu5Ac supplementation at an MOI of 10:1 for 24 h. Culture supernatants were assayed for IL-6 (n = 7), IL-12 (n = 4), TNF-α (n = 7), and IL-10 (n = 7) by ELISA. Due to donor-to-donor variation in cytokine production, cytokine levels were normalized to that of DC incubated with 35000HP grown in the absence of 1 mM Neu5Ac, which was set at 100%. Bars are means ± SDs. There were no significant differences in the levels of production of each cytokine among the groups in these assays.