Eric R. Vimr

The sialic acids ( N -acylneuraminates) are a group of nine-carbon keto-sugars existing mainly as terminal residues on animal glycoprotein and glycolipid carbohydrate chains. Bacterial commensals and pathogens exploit host sialic acids for nutrition, adhesion, or antirecognition, where N -acetyl- or N -glycolylneuraminic acids are the two predominant chemical forms of sialic acids. Each form may be modified by acetyl esters at carbon position 4, 7, 8, or 9 and by a variety of less-common modifications. Modified sialic acids produce challenges for colonizing bacteria, because the chemical alterations to N -acetylneuraminic acid (Neu5Ac) confer increased resistance to sialidase and aldolase activities essential for the catabolism of host sialic acids. Bacteria with O -acetyl sialate esterase(s) utilize acetylated sialic acids for growth, thereby gaining a presumed metabolic advantage over competitors lacking this activity. Here, we demonstrate the esterase activity of Escherichia coli NanS after purifying it as a C-terminal HaloTag fusion. Using a similar approach, we show that E. coli strain O157:H7 Stx prophage or prophage remnants invariably include paralogs of nanS often located downstream of the Shiga-like toxin genes. These paralogs may include sequences encoding N- or C-terminal domains of unknown function where the NanS domains can act as sialate O -acetyl esterases, as shown by complementation of an E. coli strain K-12 nanS mutant and the unimpaired growth of an E. coli O157 nanS mutant on O -acetylated sialic acid. We further demonstrate that nanS homologs in Streptococcus spp. also encode active esterase, demonstrating an unexpected diversity of bacterial sialate O -acetyl esterase. IMPORTANCE The sialic acids are a family of over 40 naturally occurring 9-carbon keto-sugars that function in a variety of host-bacterium interactions. These sugars occur primarily as terminal carbohydrate residues on host glycoproteins and glycolipids. Available evidence indicates that diverse bacterial species use host sialic acids for adhesion or as sources of carbon and nitrogen. Our results show that the catabolism of the diacetylated form of host sialic acid requires a specialized esterase, NanS. Our results further show that nanS homologs exist in bacteria other than Escherichia coli , as well as part of toxigenic E. coli prophage. The unexpected diversity of these enzymes suggests new avenues for investigating host-bacterium interactions. Therefore, these original results extend our previous studies of nanS to include mucosal pathogens, prophage, and prophage remnants. This expansion of the nanS superfamily suggests important, although as-yet-unknown, functions in host-microbe interactions.

NanR, one of >8,500 GntR superfamily helix-turn-helix transcriptional regulators, controls expression of the genes required for catabolism of sialic acids in Escherichia coli . It is predicted to do the same in related bacteria harboring orthologs of nanR . The sialic acids are a family of over 40 naturally occurring nine-carbon keto-sugar acids found mainly in the animal lineage, which includes starfish to humans in the deuterostome lineage. Sialic acids function in development, immunity, protein localization and stability, and homeostasis. They also serve as microbial carbon and nitrogen sources and ligands for cell recognition during host colonization. The importance of microbial sialic acid metabolism for host-microbe interactions has made it a target for therapeutic development. Exploiting this target depends on understanding sialometabolic pathways in a wide range of evolutionarily distinct bacteria. Here, we show by transcriptome, genetic, and biochemical analyses that the most common sialic acid, N -acetylneuraminate, induces the nanATEK-yhcH , yjhATS ( nanCMS ), and yjhBC operons by directly inactivating NanR, converting the predominantly dimeric form of the repressor to an inactive monomer of approximately 30-kDa. Additionally, other results identify critical amino acid residues and nucleotides in the regulator and operator, respectively. The combined results better define how sialic acids, acting through NanR, affect the metabolic flux of an important group of host-derived metabolites. Thus, E. coli serves as a valuable model for understanding sialocatabolic pathways in bacteria.

Sialic acids are structurally diverse nine-carbon ketosugars found mostly in humans and other animals as the terminal units on carbohydrate chains linked to proteins or lipids. The sialic acids function in cell-cell and cell-molecule interactions necessary for organismic development and homeostasis. They not only pose a barrier to microorganisms inhabiting or invading an animal mucosal surface, but also present a source of potential carbon, nitrogen, and cell wall metabolites necessary for bacterial colonization, persistence, growth, and, occasionally, disease. The explosion of microbial genomic sequencing projects reveals remarkable diversity in bacterial sialic acid metabolic potential. How bacteria exploit host sialic acids includes a surprisingly complex array of metabolic and regulatory capabilities that is just now entering a mature research stage. This paper attempts to describe the variety of bacterial sialometabolic systems by focusing on recent advances at the molecular and host-microbe-interaction levels. The hope is that this focus will provide a framework for further research that holds promise for better understanding of the metabolic interplay between bacterial growth and the host environment. An ability to modify or block this interplay has already yielded important new insights into potentially new therapeutic approaches for modifying or blocking bacterial colonization or infection.

Polysialic acid capsules are the major virulence factors in Escherichia coliK1, K92, and groups B and C meningococci. The sialic acid monomers (2-keto-3-deoxy-5-acetamido-7,8,9-d-glycero-d-galacto-nonulosonic acids) comprising these homopolymeric polysaccharide chains can be selectively modified with 1,2-diamino-4,5-methylenedioxy-benzene to produce highly fluorescent quinoxalinone derivatives distinguished by their elution times during reverse phase chromatography. Here, we describe methods to release the constituent capsular polysialic acid monomers, and to detect and quantify them by sensitive fluorometry. There are relatively few 2-keto acids in bacteria, making it possible to rapidly analyze samples even without prior purification of capsular polysaccharides.

The nanATEK-yhcH, yjhATS, and yjhBC operons in Escherichia coli are coregulated by environmental N-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl N-acetylneuraminic acid esterase required for E. coli to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites.

The outer membrane (OM) of almost all Gram-negative bacteria is composed of phospholipids, lipopolysaccharide, proteins and capsular or loosely adherent polysaccharides that together mediate cellular interactions with diverse environments. Most OM components are synthesized intracellularly or at the inner membrane (IM) and thus require an export mechanism. This mini-review focuses on recent progress in understanding how synthesis of one kind of capsular polysaccharide (group 2) is coupled to the export apparatus located in the IM and spanning the periplasmic space, thus providing a transport channel to the cell surface. Although the model system for these investigations is the medically important extraintestinal pathogenEscherichia coliK1 and its polysialic acid capsule, the conclusions are general for other group 2 and group 2-like polysaccharides synthesized by many different bacterial species.