Structural insights into the regulation of sialic acid catabolism by the Vibrio vulnificus transcriptional repressor NanR

Abstract

Pathogenic and commensal bacteria that experience limited nutrient availability in their host have evolved sophisticated systems to catabolize the mucin sugar N-acetylneuraminic acid, thereby facilitating their survival and colonization. The correct function of the associated catabolic machinery is particularly crucial for the pathogenesis of enteropathogenic bacteria during infection, although the molecular mechanisms involved with the regulation of the catabolic machinery are unknown. This study reports the complex structure of NanR, a repressor of the N-acetylneuraminate (nan) genes responsible for N-acetylneuraminic acid catabolism, and its regulatory ligand, N-acetylmannosamine 6-phosphate (ManNAc-6P), in the human pathogenic bacterium Vibrio vulnificus. Structural studies combined with electron microscopic, biochemical, and in vivo analysis demonstrated that NanR forms a dimer in which the two monomers create an arched tunnel-like DNA-binding space, which contains positively charged residues that interact with the nan promoter. The interaction between the NanR dimer and DNA is alleviated by the ManNAc-6P-mediated relocation of residues in the ligand-binding domain of NanR, which subsequently relieves the repressive effect of NanR and induces the transcription of the nan genes. Survival studies in which mice were challenged with a ManNAc-6P-binding-defective mutant strain of V. vulnificus demonstrated that this relocation of NanR residues is critical for V. vulnificus pathogenesis. In summary, this study presents a model of the mechanism that regulates sialic acid catabolism via NanR in V. vulnificus.nan gene repressor | mucin sugar utilization P athogenic bacteria that colonize the host gut are exposed to an adverse environment and competitors (1, 2). These bacteria overcome the limited availability of nutrients in the gut (3) by using alternative carbon sources (4). The mammalian intestinal tract is protected by a mucus layer, which contains heavily glycosylated mucin proteins that comprise up to 85% carbohydrate (5, 6). Sialic acids, which can be used as an energy source by a variety of microbial pathogens and commensals, are found at the distal ends of the mucin carbohydrate chains (7,8). Because the most abundant sialic acid is N-acetylneuraminic acid (Neu5Ac) (7-9), intestinal commensal and pathogenic bacteria have most likely evolved elaborate systems for the catabolic utilization of this substrate.Escherichia coli, Vibrio cholerae, Vibrio vulnificus, and Staphylococcus aureus can grow by using Neu5Ac as a sole carbon source (10-13), and the N-acetylneuraminate (nan) genes responsible for Neu5Ac utilization are up-regulated during their growth on mucus or in the mammalian intestine (3,14). The colonization and pathogenic activities of these bacteria are affected severely by mutations in the nan genes (3, 12, 15). This phenotype suggests that the correct expression and function of the proteins responsible for Neu5Ac catabolism are essential for the growth and survival of enteric bacteria, although onl...

Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR

“…In contrast to the GntRtype regulators of genes for Neu5Ac utilization from E. coli and B. breve, which both depend on the presence of Neu5Ac (45,46) as an effector, binding of C. glutamicum NanR to its target promoters was relieved in the presence of 0.6 mM GlcNAc-6P and at slightly higher concentrations of ManNAc-6P. Control by intermediates of Neu5Ac degradation has also been reported for the RipR-type regulators of Neu5Ac metabolism from V. vulnificus, S. pneumoniae, S. aureus, and H. influenzae, which are activated in the presence of ManNAc-6P, ManNAc, and GlcN-6P, respectively (39,42,72,73) (see Table S1 in the supplemental material). Thus, NanR of C. glutamicum is the first transcriptional regulator of sialic acid metabolism depending on the inducer GlcNAc-6P and the first of the GntR-type regulators independent of Neu5Ac (see Table S1).…”

“…In H. influenzae, transcription of the nan and the siaPT operons for Neu5Ac utilization is repressed by SiaR and is activated in the presence of glucosamine 6-phosphate (see Table S1 in the supplemental material), which is an intermediate of Neu5Ac catabolism and acts as a coactivator of the RpiR-type regulator SiaR (37,(39)(40)(41). The RpiRtype transcriptional repressor NanR of V. vulnificus controls nanT PSI AR (for the triapartite ATP-independent transporter, Neu5Ac aldolase, and the nan gene repressor), nanEK, and the nagA operons (42,43). Binding of the Neu5Ac catabolism intermediate N-acetylmannosamine-6-phosphate (ManNAc-6P) to V. vulnificus NanR mediates relocation of residues in the ligand binding site, thus alleviating the interaction between the NanR dimer and DNA and subsequently relieving the repression by NanR and inducing transcription of the nan operons (42) (see Table S1).…”

IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells

“…Mortalities of mice infected with the wild-type and iscR mutant strains were compared as described elsewhere (26). Groups of (n ϭ 20) 7-weekold ICR female mice (specific-pathogen-free; Seoul National University) were starved without food and water for 12 h until infection.…”

Structural and Functional Characterization of the Clostridium perfringens N-Acetylmannosamine-6-phosphate 2-Epimerase Essential for the Sialic Acid Salvage Pathway

“…Sialic acids are especially abundant in the intestinal tract, where they represent a major constituent of mucins and play a role in cell wall synthesis and pathogenesis (5)(6)(7). Utilization of sialic acid as a carbon source for growth has been demonstrated for the pathogenetic Staphylococcus aureus bacteria (8) as well as Streptococcus pneumoniae (9), and the importance of sialic acid catabolism in pathogenic bacteria has been documented for the foodborne enteropathogen Vibrio vulnificus (10).…”