Broad-Spectrum Legionaminic Acid-Specific Antibodies in Pooled Human IgGs Revealed by Glycan Microarrays with Chemoenzymatically Synthesized Nonulosonosides

The presence and the level of antibodies in human sera against bacterial glycans are indications of prior encounters with similar antigens and/or the bacteria that express them by the immune system. An increasing number of pathogenic bacteria that cause human diseases have been shown to express polysaccharides containing a bacterial nonulosonic acid called 5,7-di-N-acetyllegionaminic acid (Leg5,7Ac2). To investigate the immune recognition of Leg5,7Ac2, which is critical for the fight against bacterial infections, a highly effective chemoenzymatic synthon strategy was applied to construct a library of α2–3/6-linked Leg5,7Ac2-glycans via their diazido-derivatives (Leg5,7diN3-glycans) formed by efficient one-pot three-enzyme (OP3E) synthetic systems from a diazido-derivative of a six-carbon monosaccharide precursor. Glycan microarray studies using this synthetic library of a Leg5,7Ac2-capped collection of diverse underlying glycan carriers and their matched sialoside counterparts revealed specific recognition of Leg5,7Ac2 by human IgG antibodies pooled from thousands of healthy donors (IVIG), suggesting prior human encounters with Leg5,7Ac2-expressing pathogenic bacteria at the population level. These biologically relevant Leg5,7Ac2-glycans and their immune recognition assays are important tools to begin elucidating their biological roles, particularly in the context of infection and host–pathogen interactions.


Introduction
Human sera contain protective antibodies against a large range of carbohydrate epitopes expressed by different species [1][2][3][4].These antibodies are developed either naturally or in response to infections, vaccinations, or other exposures to antigens.Human antibodies recognizing bacterial glycans have been revealed by profiling human sera and pooled human immunoglobulins against bacterial surface carbohydrate antigens immobilized on glycan microarrays [5].Synthetic glycans with defined structures are important probes for these studies.Access to such synthetic glycans, especially those with complex structures that are challenging to obtain either by purification from natural sources or by chemical synthesis, is greatly facilitated by the development of efficient chemoenzymatic methods.Glycans and glycoconjugates containing nine-carbon α-keto acids called nonulosonic acids (NulOs) [6] are among the challenging synthetic targets [7].The most well-known NulOs are sialic acids (Sias) based on a neuraminic acid (Neu) or a 2-keto-3-deoxy-nononic acid (Kdn) backbone in the deuterostome lineage of animals and in certain bacteria.In vertebrates, Sias are often presented on the termini of glycans on cell surface glycoconjugates and N-acetylneuraminic acid (Neu5Ac, 1, Figure 1) is the most common and well-studied sialic acid form.Bacterial nonulosonic acid (NulO) 5,7-di-N-acetyllegionaminic acid (Leg5,7Ac 2 , 2) (Figure 1), or 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid [6,8], is an analog of Neu5Ac (1).Leg5,7Ac 2 and Neu5Ac share an identical D-glycero-D-galacto-backbone configuration but differ for the functional groups at C7 and C9 positions.Leg5,7Ac 2 is the 7-acetamido-7,9-dideoxy derivative of Neu5Ac by replacing its C7-OH with an acetamido group and its C9-OH with a hydrogen atom (Figure 1) [9,10].
The synthesis of glycans containing Leg5,7Ac 2 and derivatives [14] by chemical methods has been challenging [15].The chemical synthetic strategies reported involved long routes, low overall yields, and a lack of stereochemical control for the formation of the biologically relevant α-legionaminic acid glycosyl linkage found in several bacterial polysaccharides [15].In addition, the obtained products were limited to monosaccharides [11,12,16] or glycosides of either monosaccharides [15,17] or disaccharides [18,19].
Sialic acids are known to belong to 'self-associated molecular patterns' (SAMPs) that provide a protective shield for glycoconjugates and cells [47], including from circulating antibodies [46].To investigate human IgG immune recognition of Leg5,7Ac 2 within the context of 'pathogen-associated molecular patterns' (PAMPs), enzymatic cleavage of these sialic acids analogs on the arrays was evaluated by two sialidases, Arthrobacter ureafaciens sialidase (AUS) and Clostridium perfringens neuraminidase (NCP), that peel-off terminal sialic acid moieties from glycans printed on the array.Both sialidases effectively removed Neu5Ac and Neu5Gc, exposing the underlying de-capped glycans to a robust binding of IVIG anti-glycan antibodies.However, all Leg5,7Ac 2 -glycans and Neu5Ac7NAc-glycans were found to be resistant to such cleavage by these two bacterial sialidases (Figure 3a), which correlated well with our previous results [9,48].Despite the close similarity between Neu5Ac and Leg5,7Ac 2 , the glycans capped with the latter become resistant to sialidase cleavage, potentially providing an immunological advantage to a human invading pathogen that would be coated with such PAMP glycans.This is further supported by recent studies of Campylobacter jejuni, the leading cause of foodborne bacterial gastroenteritis, revealing that expression of legionaminic acid on its bacterial flagellin serves as a virulence factor supporting enhanced biofilm formation, auto-agglutination, and host colonization [24,49].On the other hand, pathogens also commonly use SAMPs as a molecular mimicry mechanism to evade host immunity, particularly by sialic acid sugar coating (e.g., on lipooligosaccharide; LOS) using sialyltransferases that they express.LOS sialylation shields bacteria from complement and cationic peptides and also engages inhibitory siglec receptors on host immune cells to dampen inflammatory responses [50,51].Unlike the molecular mimicry properties of Neu5Ac used by pathogenic bacteria such as Neisseria gonorrhoeae, which causes sexually transmitted gonorrhea infection, incorporating Leg5,7Ac 2 instead of Neu5Ac to the LOS by Neisseria gonorrhoeae had been found to make the bacterium sensitive to complement and human serum [51,52].Therefore, applying CMP-Leg5,7Ac 2 , which can be used by some human pathogens to decorate their LOS with Leg5,7Ac 2 , has been proposed as a novel therapeutic approach to address the global threat of multidrug-resistant bacteria [51][52][53].Nevertheless, the host-pathogen interplay involving sialic acids and other nonulosonic acids with sialidases, siglecs, complement, cationic antimicrobial peptides, serum antibodies, and the complement system is complex and warrants further investigation.To gain further insight into human IgG immune recognition of Leg5,7Ac 2 , binding of IVIG to Leg5,7Ac 2 -glycans on the glycan microarray was examined in the presence of selected competing glycans, including methyl α-N-glycolylneuraminoside (2-Omethyl-αNeu5Gc, or Gc2Me), glycopeptides of diverse N/O-glycans produced from mice sera digestion (mGP) [46], and the glycans that showed top preference of binding on the arrays including glycans ID-51, ID-502, ID-508 (Core1β; Leg5,7Ac 2 α3Core1β; and Leg5,7Ac 2 α6Core1β, respectively; Figure 3a).This analysis revealed that only αNeu5Gc (Gc2Me) did not affect IVIG binding to Leg5,7Ac 2 -glycans, while all other glycans inhibited its binding (Figure 3b).Particularly, while IVIG binding to all Leg5,7Ac 2 -glycans was inhibited by 50% with Core1β (ID-51) and by 80% with mGP, the competing Leg5,7Ac 2 α3/6Core1βglycans (ID-502/508) completely abolished it (Figure 3b,c).This suggests that human IgG is enriched with antibodies that recognize diverse Leg5,7Ac 2 -glycans, in which the Leg5,7Ac 2 moiety is critical for their recognition, although with some promiscuity with the variation in the underlying glycans including Core1/Lactose/LacNAc. Based on these inhibition studies together with previous structural studies of antibodies against sialyl-Tn [54] and sialyl Lewis a (sLe a ) [55], it is plausible that the Leg5,7Ac 2 moiety is buried within the binding pocket of these anti-Leg5,7Ac 2 IgG antibodies.

General Procedures for Converting Cbz-Tagged Glycans to Glycosyl Propylamines
To a stirring solution of a Cbz-tagged glycoside selected from 25-37 (5-10 mg) in watermethanol solution (3 mL, 1:2 by volume), a catalytic amount of 10% palladium on charcoal was added to a 50 mL round bottom flask.The mixture was stirred under a hydrogen environment for 2-5 h.The solution was passed through a syringe filter.Methanol was removed from the filtrate by blowing air inside a fume hood.The residue was lyophilized and used directly for microarray assays.

Glycan Microarray Binding Assays
Slides were developed and analyzed as previously described [58] with some modifications.Slides were rehydrated with dH 2 O and incubated for 30 min in a staining dish with 50 • C pre-warmed ethanolamine (0.05 M) in Tris-HCl (0.1 M, pH 9.0), then washed with 50 • C pre-warmed dH 2 O. Slides were centrifuged at 200× g for five min then fitted with a ProPlate™ Multi-Array 16-well slide module (Grace Bio-lab) to divide into the sub-arrays (blocks).Slides were washed with PBST (0.1% Tween 20), aspirated, and blocked with 200 µL/sub-array of blocking buffer (PBS pH 7.3 + 1% w/v ovalbumin) for 1 h at RT with gentle shaking.Next, the blocking solution was aspirated and a 100 µL/sub-array of detection lectins/antibodies diluted in blocking buffer (lectins binding buffer also included divalent cations, as detailed in the ESI glycan microarray data file in Supplementary Materials) were incubated with gentle shaking for 2 h at RT.
Binding was detected with Cy3-labeled secondary detection reagent diluted in PBS at 200 µL/sub-array at RT for 1 h with gentle shaking (0.4 ng/µL Cy3 AffiniPure goat-antihuman IgG (H + L), 0.375 ng/µL Cy3-anti-chicken IgY, 1.2 ng/µL Cy3-sterptavidin).Slides were washed 4 times with PBST, then with PBS (without Tween-20) for 10 min followed by removal from ProPlate™ Multi-Array slide module and immediately dipping in a staining dish with dH 2 O for 10 min with shaking.Slides were then centrifuged at 200× g for 5 min and the dry slides were immediately scanned.

Array Slide Processing
Processed slides were scanned and analyzed as described [45,58] at 10 µm resolution with a Genepix 4000B microarray scanner (Molecular Devices, San Jose, CA, USA) using 350 gain.Image analysis was carried out with Genepix Pro 7.0 analysis software (Molecular Devices).Spots were defined as circular features with a variable radius as determined by the Genepix scanning software.Local background subtraction was performed.RFU from each spot was calculated.

Statistics
Statistical analyses were performed using GraphPad Prism 10.2.0 and described in context in the Figure legends.

Conclusions
We demonstrate here that the chemoenzymatic synthon strategy using 6deoxyMan2,4diN 3 (4) as the starting material in OP3E sialylation systems is highly efficient in constructing a comprehensive library of α2-3and α2-6-linked Leg5,7Ac 2 -glycosides via the enzymatic formation of Leg5,7diN 3 , CMP-Leg5,7diN 3 , and Leg5,7diN 3 -glycosides intermediates, followed by chemical conversion of the azido to the acetamido groups.The hydrophobic UV-detectable Cbz group in the sialyltransferase acceptors facilitated product purification from the OP3E systems.Removal of the Cbz group in the ProNHCbz-tagged Leg5,7Ac 2 -glycosides produced ProNH 2 -tagged Leg5,7Ac 2 -glycosides ready for immobilization on the activated glass slide surface.Glycan microarray studies using the Leg5,7Ac 2 -glycosides and their structurally related sialoside pairs revealed diverse human IgG antibodies that selectively recognize the α-linked bacterial nonulosonic acid Leg5,7Ac 2 .Such broad-spectrum recognition by human IgG antibodies pooled from thousands of donors (IVIG) supports previous encounters of Leg5,7Ac 2 -expressing pathogens by the general human population.Overall, these resources and analytical tools are important tools for further in-depth investigation of Leg5,7Ac 2involved bacterial physiology, virulence, pathogenicity, and host-pathogen interactions.