The Neisseria meningitidis ZnuD Zinc Receptor Contributes to Interactions with Epithelial Cells and Supports Heme Utilization when Expressed in Escherichia coli

Abstract

Neisseria meningitidis employs redundant heme acquisition mechanisms, including TonB receptor-dependent and receptorindependent uptakes. The TonB-dependent zinc receptor ZnuD shares significant sequence similarity to HumA, a heme receptor of Moraxella catarrhalis, and contains conserved motifs found in many heme utilization proteins. We present data showing that, when expressed in Escherichia coli, ZnuD allowed heme capture on the cell surface and supported the heme-dependent growth of an E. coli hemA strain. Heme agarose captured ZnuD in enriched outer membrane fractions, and this binding was inhibited by excess free heme, supporting ZnuD's specific interaction with heme. However, no heme utilization defect was detected in the meningococcal znuD mutant, likely due to unknown redundant TonB-independent heme uptake mechanisms. Meningococcal replication within epithelial cells requires a functional TonB, and we found that both the znuD and tonB mutants were defective not only in survival within epithelial cells but also in adherence to and invasion of epithelial cells. Ectopic complementation rescued these phenotypes. Interestingly, while znuD expression was repressed by Zur with zinc as a cofactor, it also was induced by iron in a Zur-independent manner. A specific interaction of meningococcal Fur protein with the znuD promoter was demonstrated by electrophoretic mobility shift assay (EMSA). Thus, the meningococcal ZnuD receptor likely participates in both zinc and heme acquisition, is regulated by both Zur and Fur, and is important for meningococcal interaction with epithelial cells.

The Zinc-Responsive Regulon of Neisseria meningitidis Comprises 17 Genes under Control of a Zur Element

“…This was shown recently for znuD, whose expression also was iron induced (26). Besides its upstream Zur binding site, znuD also harbors a Fur binding site where Fur in vitro binds to, independently of Zur (26). However, we did not detect any Fur box upstream of nmb0942-nmb0941, nmb1475, nmb1497, and nmb0546, nor has znuD been regulated upon lactoferrin exposure in the study by Jordan and Saunders (23).…”

“…Therefore, regulation of these genes upon lactoferrin exposure may additionally be accomplished by a second regulator that senses iron. This was shown recently for znuD, whose expression also was iron induced (26). Besides its upstream Zur binding site, znuD also harbors a Fur binding site where Fur in vitro binds to, independently of Zur (26).…”

“…Furthermore, znuA contributes to the in vivo pathogenicity of Salmonella enterica (3). Moreover, ZnuABC and ZnuD were lately shown to contribute to bacterial adhesion to epithelial cells in E. coli (15) and N. meningitidis (26), respectively.…”

“…Stork et al showed that expression of znuC and znuD is enhanced in a zur knockout mutant and predicted Zur binding motifs upstream of both genes based on the E. coli Zur binding sequence (59). Kumar et al subsequently demonstrated that N. meningitidis znuD is also regulated by Fur and allows for heme capture on the cell surface when expressed in E. coli (26). Yet, even for znuD, binding of Zur to the promoter of Zur-regulated genes has not been demonstrated by in vitro experiments.…”

ZnuD, a Potential Candidate for a Simple and Universal Neisseria meningitidis Vaccine

“…A putative znuCBA operon as well as putative Zur-binding sequences in regions upstream of the znuC and znuD genes were described for MenB (8). ZnuD has also been described as being regulated by iron in a Zur-independent way and to be involved in the meningococcal interaction with epithelial cells (10). Based on a limited number of sequences (n ϭ 6), ZnuD appears to be potentially well conserved.…”