Sialic Acid ( N -Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N -Acetyl Mannosamine Epimerase

Abstract

We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.Many bacteria have the ability to release sialic acids from complex glycoproteins and oligosaccharides present in the media or on cell surfaces at sites of colonization or infection. To use the released sialic acids as a rich source of carbon and nitrogen for growth, bacteria must have the ability to transport these compounds into the cell and convert the nine carbon sugars into intermediates that enter the central glycolytic pathways. The utilization of N-acetyl neuraminic acid (NANA), one of the sialic acids, has been well studied in Escherichia coli (36, 37), Haemophilus spp. (1, 35), and Clostridium spp. (38), to name a few.In many microorganisms, the genes for NANA utilization are arranged in an operon that may be regulated by a repressor protein, termed NanR. A comprehensive review of the organization and composition of several prokaryotic operons involved in NANA utilization has been published (36). Many of these operons share common components, including a transport gene for NANA (nanT), a gene encoding an aldolase (nanA) that splits NANA into pyruvate and N-acetyl mannosamine (manNAc), a gene encoding a kinase activity (nanK) that phosphorylates manNAc to form manNAc 6-P and, finally, an epimerase gene (nanE) whose product converts manNAc 6-P to N-acetylglucosamine 6-P (NAG 6-P). NAG 6-P then enters the common pathway of aminosugar utilization (21). For a schematic of the NANA utilization pathway in E. coli, the current paradigm of prokaryotic NANA utilization, see Fig. 7A.Bacteroides fragilis possesses a neuraminidase activity, which can liberate free NANA from complex glycoproteins and oligosaccharides. Go...

An Infant-associated Bacterial Commensal Utilizes Breast Milk Sialyloligosaccharides

“…13,47). Recently, B. fragilis was characterized with a peculiar Neu5Ac catabolism in which N-acetylmannosamine epimerase (pfam07221) converts manNAc to Nacetylglucosamine to be subsequently phosphorylated (48). This contrasts with the canonical pathway in which ManNAc is phopshorylated prior to epimerization (49).…”

Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections

“…Notably NanH has strong periplasmic localization signals. An extensive region of the B. fragilis chromosome (BF638R1715-BF638R1740) is devoted to sialic acid utilization and includes the nanLET operon, the nanH operon, and five stand-alone pairs of SusC/SusD homologs (21,22). Our microarray data showed that regulation of these genes was complex but the entire region was induced on day 1 following inoculation of the tissue cage, and for the most part remained elevated throughout (SI Appendix, Table S1 and Fig.…”

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

“…Second, the use of antagonists to block the binding of ManNAc-6P to NanR is unlikely to disturb the normal flora of the host because the mechanisms that regulate the nan genes in typical commensal bacteria differ from those in V. vulnificus. For example, E. coli uses Neu5Ac itself as the inducer for the transcription of the nan gene (17), whereas Bacteroides species do not generate ManNAc-6P as a catabolic intermediate of Neu5Ac (32). In summary, the elucidation of the molecular mechanisms involved with the regulation of the nan genes described in this study provides a starting point for the design of antibiotics to target life-threatening Vibrio species.…”