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Glycobiology
Glycobiology
glycob
Glycobiology
0959-6658
1460-2423
Oxford University Press

39244665
10.1093/glycob/cwae071
cwae071
Original Article
AcademicSubjects/SCI01000
Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in gram-negative bacteria
Alvarado-Melendez Erianna I Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomedical Research, Utrecht University, Universiteitsweg 99, 3584CG, Utrecht, The Netherlands

de Jong Hanna Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomedical Research, Utrecht University, Universiteitsweg 99, 3584CG, Utrecht, The Netherlands

Hartman Jet E M Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomedical Research, Utrecht University, Universiteitsweg 99, 3584CG, Utrecht, The Netherlands

Ong Jun Yang Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomedical Research, Utrecht University, Universiteitsweg 99, 3584CG, Utrecht, The Netherlands

Wösten Marc M S M Department of Biomolecular Health Sciences, Division Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584CL, Utrecht, The Netherlands

https://orcid.org/0000-0002-2368-7728
Wennekes Tom Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomedical Research, Utrecht University, Universiteitsweg 99, 3584CG, Utrecht, The Netherlands

Corresponding authors: Department of Biomolecular Health Sciences, Division Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Email: m.wosten@uu.nl (Marc M. S. M. Wösten) and Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomedical Research, Utrecht University, Utrecht, The Netherlands. Email: t.wennekes@uu.nl (Tom Wennekes)
Erianna I. Alvarado-Melendez and Hanna de Jong contributed equally.

10 2024
08 9 2024
08 9 2024
34 10 cwae07112 7 2024
27 8 2024
06 9 2024
20 9 2024
© The Author(s) 2024. Published by Oxford University Press.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

Lipooligosaccharides are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host–microbe interactions. Certain Gram-negative pathogenic bacteria cap their lipooligosaccharides with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their lipooligosaccharides. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the lipooligosaccharides of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering and Selective Exo-Enzymatic Labeling, and that together they provide tools to modify, label, detect and visualize sialylation of bacterial lipooligosaccharides.

glycoengineering
Gram-negative bacteria
lipooligosaccharides
sialic acid
sialyltransferases
European Union’s Horizon 2020 Marie Skłodowska-Curie Actions for the Innovative Training Network “Sweet Crosstalk” 814102 Dutch Research Council (NWO) via a VIDI 737.016.013 723.014.005
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pmcIntroduction

Cell surface glycoconjugates play a key role in mediating interactions between bacteria and the host (Poole et al. 2018). One important and prevalent monosaccharide found at the terminal end of several classes of host glycoconjugates is N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid. In mammals, it is found in gangliosides, N-/O-linked glycoproteins, and polysialic acid (Lewis et al. 2015), where it has important functional roles such as mediation of cell–cell interactions, modulation of the immune response, cell signaling (Varki and Gagneux 2012; Chang and Nizet 2014), but also serves as a binding site for pathogens (Hirmo et al. 1998).

Compared to mammals, Neu5Ac is less commonly found in Gram-negative bacteria. Currently, a limited number of bacterial species are known to display Neu5Ac as part of their capsular polysaccharides (CPS), or their lipopolysaccharides (LPS) and lipooligosaccharide (LOS). Some of these species can produce Neu5Ac de novo, and others have to scavenge it from the host such as Neisseria gonorrhoeae (Ghosh 2020). In particular, sialylated LOS has been found in strains of Campylobacter jejuni, Vibrio, Neisseria, and Haemophilus influenza (Dudek et al. 2022). Sialylation of bacterial LOS occurs via sialyltransferases (ST) and can result in the mimicry of host glycans, a phenomenon observed in some pathogens such as N. gonorrhoeae, Haemophilus influenzae and C. jejuni (Moran et al. 1996).

Bacteria can use this strategy of mimicking host glycans to mask their surface and avoid being recognized as foreign cells by the immune system. There are several mechanisms by which host cells distinguish between self and non-self cells. One such mechanism is the alternative complement pathway. This pathway uses factor H, surface-bound C3b and host polyanions to discriminate between cells (Pangburn et al. 2008). It is known that cells containing sialic acid do not activate this pathway (Pangburn et al. 2008). Another way of distinguishing host from foreign cells is through sialic acid binding receptors (Siglecs). Siglecs recognize sialic acid-containing ligands expressed on the surface of cells, the presence of these ligands attenuates inflammatory and innate immune responses of Toll-like receptors (Crocker et al. 2007; Paulson et al. 2012; Varki and Gagneux 2012). In this way, bacterial glycans containing Neu5Ac can be recognized by host Siglecs and factor H, which leads to dampening of immune signals or prevents the activation of the alternative complement pathway, respectively (Meri and Pangburn 1990; Kopp et al. 2012; Leaubli et al. 2022; de Jong et al. 2022a). Mimicry through sialic acid can also induce autoimmune mechanisms leading to the development of diseases like Guillain-Barré syndrome, which is caused by certain strains of C. jejuni. In the case of pathogenic Neisseria strains, sialylation of the LOS has been reported to influence the susceptibility of the bacterium to bactericidal antibodies (Rest and Frangipane 1992). This sialylation has a negative impact on the host complement pathway activation, preventing phagocytosis, reducing adhesion to neutrophils and thus also induction of an oxidative burst (Rest and Frangipane 1992; Wetzler et al. 1992; Moran et al. 1996; Carlin et al. 2009). For these reasons, the presence of Neu5Ac in bacterial LOS is considered a virulence factor, yet the precise functional role of Neu5Ac in molecular mimicry is not fully understood yet. Additionally, there may be other bacteria capable of decorating their LOS with sialic acid that have yet to be discovered. To investigate this and gain deeper insights, we need glycoengineering techniques to detect strains with active sialyltransferases and manipulate Neu5Ac in bacterial glycoconjugates.

Currently, there are several widely applied approaches to perturb and study the role of glycans in mammalian cells (Griffin and Hsieh-Wilson 2016; Nischan and Kohler 2016; Critcher et al. 2021; Edgar 2021), but only a few techniques are available to study the bacterial glycome (Calles-Garcia and Dube 2024). The most widely used approach is Metabolic Oligosaccharide Engineering (MOE). In this technique carbohydrates modified with bioorthogonal reporters are taken up by the cell, metabolically processed and transferred onto the cellular glycans. The metabolically incorporated glycan analog can be studied via its reporter by covalently attaching a fluorophore or an enrichment tag. MOE has been applied to microbes to engineer their cell wall (Sadamoto et al. 2002, 2004; Liu et al. 2009; Dumont et al. 2012; Swarts et al. 2012), image (Geva-Zatorsky et al. 2015; Hudak et al. 2017), discover glycoproteins (Besanceney-Webler et al. 2011; Champasa et al. 2013) or develop new antibacterial strategies (Kaewsapsak et al. 2013; Memmel et al. 2013; Tra and Dube 2014). Although MOE is a very useful technique, it has limitations. To modify glycans, the carbohydrate analogs used must be able to enter the cell, be metabolically processed by the native salvage pathway enzymes and finally be accepted as substrates by native intracellular glycosyltransferases. Thus, this technique cannot be applied to bacteria that lack the biosynthetic machinery to metabolically process the monosaccharide. Furthermore, shortcomings in any of these steps often result in limited control over the level and location of reporter incorporation, e.g. glycoproteins or glycolipids (Bussink et al. 2007). The metabolically generated sugar nucleotide analogs are often substrates for several related intracellular glycosyltransferases. Therefore, MOE may also result in different linkage types, e.g. α2,3- vs. α2,6-sialic acid linkages, which complicate their subsequent analysis and interpretation of their biological effects. In addition, the efficacy and precision of this technique are significantly influenced by the sugar analog employed. For instance, in mammals, UDP-GalNAc and UDP-GlcNAc can be interconverted by epimerases, which in turn are utilized by different glycosyltransferases, resulting in the incorporation of these probes on distinct glycans (Boyce et al. 2011). Another disadvantage is the toxicity associated with carbohydrate analogs containing azido moieties and Cu(I), when copper-catalyzed azide-alkyne cycloaddition (CuAAC) is used (Han et al. 2018; Liu et al. 2022; Saïdi et al. 2022).

Another glycoengineering technique is selective exoenzymatic labeling (SEEL) (Sun et al. 2016; Yu et al. 2016) that we have applied to label the LOS of N. gonorrhoeae (de Jong et al. 2022b). SEEL uses an externally applied recombinant glycosyltransferase to selectively label the outside of a cell with tailor-made sugar nucleotide analogs. The advantages of SEEL are the precise incorporation of a monosaccharide derivative onto a defined acceptor on the cell surface with a known linkage type, and low cytotoxicity. The established linkage type can also be non-native to the bacterium, such as α-2,6-Neu5Ac instead of the naturally occurring α-2,3-Neu5Ac on N. gonorrhoeae (Gilbert et al. 1996), which is a unique feature of SEEL compared to other glycoengineering techniques. One disadvantage of this technique is the need for a specific recombinant sialyltransferase that can recognize the LOS epitope in the bacterium of interest. For several bacteria the LOS structures are still not known. When applying SEEL to N. gonorrhoeae (de Jong et al. 2022b), we observed that wild-type bacteria were able to incorporate the sugar nucleotide analogs with their own sialyltransferases. N. gonorrhoeae was previously reported to scavenge sialic acid nucleotides from the environment and transfer them to its lipooligosaccharides via native sialyltransferases (Nairn et al. 1988; Gulati et al. 2020, 2015; Jen et al. 2021). In our experiments, we also noticed that the level of sialylation achieved when using SEEL in N. gonorrhoeae was lower than the produced by the bacterium native sialyltransferases (de Jong et al. 2022b). The incorporation of fluorescently labeled sialic acid onto cellular glycans using endogenous sialyltransferases has been previously reported in zebrafish embryos and has proven to be an effective approach for direct live visualization of a dynamic system (Honk et al. 2019).

Intrigued by the possibility of using endogenous enzymes to engineer and visualize cell surface glycoconjugates of bacteria, we sought to further investigate this labeling strategy on two other pathogens known for displaying sialylated LOS (Scheme 1). We selected Non-typeable H. influenzae (NTHi), and C. jejuni (strains associated with Guillain-Barré syndrome), to assess the labeling of their own LOS using their native sialyltransferases (NSTs) and CMP-Neu5Ac analogs. We performed a qualitative comparison between this approach, MOE and SEEL. To further extend the application of glycoengineering via NSTs to more bacteria, we performed a BLAST search using as input the annotated sialyltransferases found in N. gonorrhoeae (Lst), C. jejuni (CstII) and NTHi (Lic3A, LsgB and SiaA). From the BLAST search, we selected 49 strains of Gram-negative bacteria that includes relevant human pathogens, commensal, and zoonotic species. We applied our labeling strategy to label the lipooligosaccharides and detect native sialyltransferase activity in this selection of Gram-negative bacteria with Neu5Ac analogs. This revealed that a variety of pathogenic and commensal bacteria can utilize CMP-Neu5Ac analogs to label their LOS. For some of the bacteria, this represents the first reported evidence of sialyltransferase activity. It also showcased the ease by which bacterial native sialyltransferase activity and sialylated surface glycans can be detected via this approach.

Scheme 1 Schematic overview of this work.

Results and discussion

Comparing bacterial glycoengineering via NSTs, MOE and SEEL

For its glycan mimicry (de Jong et al. 2022b), N. gonorrhoeae transfers scavenged CMP-Neu5Ac onto its LOS with an α-2,3 linkage with its sialyltransferase Lst (Mubaiwa et al. 2017). As N. gonorrhoeae lacks the genes for the biosynthesis of CMP-Neu5Ac, MOE is not possible (Fig. 1A). This was verified by probing both the wild type N. gonorrhoeae and a sialyltransferase mutant (Δlst) with known MOE probe, Neu5Az, followed by a Strain-Promoted Alkyne-Azide Cycloaddition reaction (SPAAC) click reaction and observing no labeling of the LOS (Fig. 1A). We previously showed that N. gonorrhoeae LOS can be labelled using SEEL (de Jong et al. 2022b). We also found that endogenous sialyltransferases are able to transfer CMP-Neu5Az onto their LOS, something that has also been observed by other groups (Gulati et al. 2020, 2015; Jen et al. 2021). We first wanted to further investigate the substrate scope of the Lst native sialyltransferase and the speed of the NST approach. For this we exposed N. gonorrhoeae to two different analogs of the sugar nucleotide donor: the known CMP-Neu5Az (Fig. 1B), and the not yet tested CMP-Neu5biotin (Fig. 1C) and observed that LOS labeling using CMP-Neu5Az occurs quickly, within minutes, after incubation with the probe (Fig. 2D). Unsurprisingly, we also found that CMP-Neu5biotin was incorporated in the LOS, showing that larger modifications at the fifth position are accepted by Lst, as was already observed in our previous work when using CMP-Neu5AF488 (de Jong et al. 2022b).

Fig. 1 MOE and NSTs applied to Neisseria gonorrhoeae. A) MOE with Neu5Az on N. gonorrhoeae wild type and the ST mutant (Δlst) followed by a SPAAC with DBCO-PEG4-biotin. B) Labeling of N. gonorrhoeae WT LOS by NSTs followed by SPAAC using DBCO-PEG4-biotin. C) Incorporation of Neu5biotin into the Neisserial LOS by NSTs. LOS was analyzed on western blot and by silver staining. D) Incubation of N. gonorrhoeae with CMP-Neu5Az at different time points followed by SPAAC click with DBCO-PEG4-biotin. E) Serum resistance of N. gonorrhoeae which was grown without or with a nucleotide sugar; CMP-Neu5Ac or CMP-Neu5Az. Colony forming units were counted after incubation in the presence of normal human serum (NHS) or heat inactivated NHS (HI-NHS), and reported as % survival.

Fig. 2 A) MOE applied to label LOS of NTHi wildtype (WT), and a sialic acid transporter mutant (ΔsiaP) using Neu5Az followed by SPAAC reaction with DBCO-PEG4-biotin. B) Labeling of LOS by NSTs of NTHi using CMP-Neu5Az, followed by SPAAC with DBCO-PEG4-biotin. C) Labeling of NTHi LOS by the NSTs using a concentration range of CMP-Neu5biotin.

Additionally, to show the utility of this labeling method to study the effect of bacterial sialylation on serum-mediated killing we performed an assay using the CMP-Neu5Az. Gulati et al., previously showed that N. gonorrhoeae has altered resistance against serum-mediated killing when the bacteria were modified with other sialic acids than Neu5Ac, such as legionaminic acid (Gulati et al. 2020, 2015). However, sialylation with Neu5Az has not been reported yet. In a serum-resistance assay with bacteria covered by Neu5Az, the bacteria were less resistant against serum compared to bacteria treated with native CMP-Neu5Ac (Fig. 1E). This observation fits the trend that a small modification on the glycan can perturb the interaction with factors from serum, which also has been seen for the 9-azido derivative of the nucleotide sugar (Gulati et al. 2015).

The next Gram-negative bacterium we investigated, non-typeable H. influenzae (NTHi), is known for displaying sialic acid on its LOS to increase resistance to serum-mediated killing (Apicella 2012; Heise et al. 2018; de Jong et al. 2022a). We started our investigation by corroborating the results obtained by Heise et al. when applying MOE to NTHi (Heise et al. 2018). We found that Neu5Az was incorporated into its LOS and not by a mutant lacking the sialic acid transporter SiaP (Fig. 2A). We did not observe incorporation of Neu5Az into other cell surface glycoproteins (Supporting information, Fig. S1), but did notice a decrease in bacterial growth, indicative of cell toxicity, when using a high concentration of Neu5Az (1 mM) (Supporting information, Fig. S2).

We next tested SEEL to label NTHi using CMP-Neu5biotin in combination with sialyltransferases of mammalian origin first (ST6Gal1) and then STs from bacteria (Pmst1, Pmst3 and Pd2,3). We did not observe any labeling of NTHi LOS, with the different enzymes tested, which indicated that to achieve labeling via the SEEL technique, the selection of STs is essential, and the method is compromised for bacteria in which the LOS acceptor structure or LOS shielding is unknown (Supporting information, Fig. S3). We however did observe that in the absence of exogenous enzyme, the NSTs from NTHi can incorporate the unnatural Neu5Ac analog into the LOS (Fig. 2B). We tested the ability of the native sialyltransferases using CMP-Neu5Az, and CMP-Neu5biotin, at different concentrations. With both nucleotide sugars we observed labeling of the LOS. When using CMP-Neu5biotin we observed labeling up to a concentration as low as 0.8 μM (Fig. 2C).

We continued our investigation with C. jejuni, the major cause of bacterial gastroenteritis in the developing world. Certain C. jejuni strains are also linked to the autoimmune disease Guillain-Barré syndrome (GBS) and have unique LOS structures that mimic the terminal epitopes of human glycans. C. jejuni’s LOS can be sialylated and bind to host receptors, such as Siglec 1 and 7 (Heikema et al. 2013b). This mimicry can however also lead to the production of anti-ganglioside antibodies targeting the nerves of the host during the disease, which can manifest in a variety of ways, including symptoms such as mild paralysis to acute pain (Yuki et al. 2004; Van Den Berg et al. 2014). Due to the link between the sialylated LOS of C. jejuni and the development of GBS, this is an intriguing target to manipulate and study. LOS sialylation occurs via sialyltransferases CstI and CstII that create either α-2,3- or both α-2,3- and α-2,8-linkages depending on a point mutation (Gilbert et al. 2002; Godschalk et al. 2007). In our study we focused on two strains of C. jejuni that are associated with GBS: GB11 and GB19, along with the corresponding mutant strains of CstII (Godschalk et al. 2007, 2004; Louwen et al. 2008).

We first tested MOE by using Neu5Az to attempt labeling C. jejuni’s LOS. For the WT strains we observed labeling and as expected not for the sialyltransferase mutants (Fig. 3A). This result is the first properly documented example of MOE in C. jejuni. Next, SEEL was investigated in the CstII deficient mutants of all three strains using recombinant CstII as the external sialyltransferase. The ST, CstII, has been characterized as a bifunctional sialyltransferase having both α2,3-sialyltransferase and α2,8-sialyltransferase activities, with a preference for α2,3-sialosides substrates (Babulic et al. 2023). We found that under our experimental conditions, CstII can sialylate the LOS of strain GB11, but not the LOS of strain GB19 (Supporting information, Fig. S4). We hypothesize that, since the LOS structure of these strains differ, strain GB19 probably requires another sialyltransferase. We therefore tested SEEL on C. jejuni GB19 using a combination of CstII and CstI (Chiu et al. 2004), and labeling was indeed observed (Supporting information, Fig. S5). With SEEL in these GB Campylobacter strains proving successful, we tested the ability of the native STs in the WT strains to transfer the nucleotide sugar derivative. Figures 3B-C show successful labeling of the LOS using CMP-Neu5Az, CMP-Neu5biotin and with a fluorescent reporter group (CMP-Neu5AF488). No labeling was observed for the strains lacking the cstII genes, as we demonstrated with the CstII deficient mutants (ΔcstII) of the GB strains 11 and 19.

Fig. 3 MOE, SEEL and native sialyltransferases, applied to C. jejuni. A) MOE with Neu5Az on GB19 wildtype and the CstII sialyltransferase mutant. The probe is incorporated into the LOS of the WT, as observed after a SPAAC reaction with DBCO-PEG4-biotin. B) SEEL with exogenous applied CstII. C) Labeling of LOS by the native sialyltransferases using CMP-Neu5biotin. D) In-gel fluorescence of LOS modified by native sialyltransferases with CMP-Neu5AF488.

After having established that strains of NTHi, N. gonorrhoeae and C. jejuni could incorporate modified nucleotide sugars via their own native sialyltransferases, we sought to investigate whether other bacterial species were also capable of this activity and to develop a strategy to use CMP-Neu5Ac analogs to scan bacteria for the expression of active STs and the capacity to use exogeneous CMP-Neu5Ac to sialylate their LOS.

Scanning for sialyltransferase activity and sialylated LOS in other pathogenic bacteria

We started with a BLAST search on the six sialyltransferase genes known or expected to be capable of ST activity in NTHi, N. gonorrhoeae and C. jejuni: Lst (α2,3-ST, from N. gonorrhoeae strain F62), Lic3A (α2,3-ST, from NTHi R2886), SiaA (α2,8- ST, from NTHi), LsgB from NTHi, CstII (α2,3-ST, C. jejuni) and PmST1 (α2,3-ST from Pasteurella multocida pm70) (see Supporting information, Table S2). From the hits of this BLAST search, we have selected 49 Gram-negative bacteria strains, among them human pathogens, commensal and zoonotic species. This list includes representatives of the Campylobacter genus, such as C. insulaenigreae, Campylobacter upsaliensis, Campylobacter lari and Campylobacter coli; and Pasteurella species such as Pasteurella dagmatis and P. multocida. We also included Neisseria meningitidis strains, a variety of Haemophilus strains, Prevotella timonensis and Prevotella bivia species and commensal bacteria such as Neisseria lactamica and Haemophilus parainfluenzae, to make the selection as diverse as possible.

The activity of the sialyltransferases of these bacteria was tested using our previously optimized standardized conditions, developed during the LOS labeling of NTHi, N. gonorrhoeae and C. jejuni. We however do not exclude the possibility that the sialyltransferase activity of these bacteria might alter under different labeling, environmental or culturing conditions. In all the experiments, we examined the labeling of cell surface glycoconjugates such as LPS/LOS, and also the possible incorporation of Neu5Az into cell-surface glycoproteins and capsular polysaccharides (CPS) in those bacteria that produce them.

We used a two-step labeling approach to label bacterial LOS by incubating the bacteria with CMP-Neu5Az, followed by a SPAAC reaction using DBCO-PEG4-biotin. For some experiments we also use compared this to CuAAC for the click reaction and obtained identical results (Supporting information, Fig. S6). To visualize the potentially labeled LOS, samples were digested with Proteinase K overnight, and boiled in loading buffer before loading onto tris-tricine gels. It is worth mentioning that most of the LOS structures labelled in the experiments reported here have not yet been characterized or reported in the literature, so we could not directly relate the results to the expected size of the LOS.

We observed labeling of the LOS/LPS in 39 out of 49 selected bacteria. Among these: P. timonensis, Haemophilus aegyptius, H. parainfluenzae, C. upsaliensis, Campylobacter insulaenigrae, P. multocida, P. dagmatis, N. lactamica, and N. meningitidis. These results indicate that these bacteria are expressing active STs capable of utilizing exogenous CMP-Neu5Az to label their LOS. However, it should be emphasized that the absence of labeling in some experiments does not exclude the presence of sialyltransferases in those bacteria.

Protein sialylation was evaluated by running samples on SDS gels without the Proteinase K digestion step. We also assessed the labeling of CPS of strains C. jejuni GB11 and GB19, N. gonorrhoeae and N. meningitidis B, C and W135; we did not observe labeling of proteins or CPS for any of the bacteria when incubated with CMP-Neu5Az (Supporting information, Fig. S7 and S8).

Sialylation via the method we employ requires an active sialyltransferase, a substrate CMP-Neu5Az, and an appropriate acceptor epitope in the LOS. Our experiments represent a situation for these bacteria where they are in a simulated environment rich in their ST sugar-nucleotide substrate. The detection of sialyltransferase activity via our method hints at a mechanism by which these bacteria can use CMP-Neu5Ac available from the environment to cap their LOS/LPS. The short incubation time required for labeling, as seen in N. gonorrhoeae (Fig. 1D), indicates that the STs could be located at or near the bacterial surface (Shell et al. 2002). Currently, the exact localization of the Neu5Az within the labelled LOS is also unknown, but we hypothesize that it is located at a readily accessible terminal position of the LOS, given the rapid labeling observed, but we intend to further investigate this in future studies.

It is known that the source of Neu5Ac for N. gonorrhoeae originates from human erythrocytes (Nairn et al. 1988). Recent studies have shown that platelets are a source of nucleotide sugars, that can be released after a stress trigger, and that sialylation via this process is possible in vivo (Lee et al. 2014; Jones et al. 2016; Manhardt et al. 2017). Platelets or other sources such as liberation in co-culture with other bacteria, are a plausible source of nucleotide sugars. An alternative mechanism for the incorporation of nucleotide sugars could be the uptake of these nucleotide sugars from the environment and sialyltransferase activity in the cytoplasm instead of on the surface. From our data it is not possible to draw definite conclusions about the exact location of the sialyltransferases. We previously reported microscopy data for N. gonorrhoeae incubated with CMP-Neu5AF488, which showed no fluorescence in the cytoplasm (de Jong et al. 2022b). Since the fluorescence signal was not distributed throughout the cytoplasm and was localized outside the membrane, this tentatively indicates that the sialyltransferases are active in the periplasm or on the surface. In addition, we here fluorescently labeled another strain, N. meningitidis B with CMP-Neu5AF488, and observed a similar localization (See Fig. 4). We excluded the possibility that the hydrolysis of the sugar nucleotides provides sialic acid analogs that are then metabolized and incorporated into the LOS, since LOS labeling was observed almost immediately after incubation with CMP-Neu5Az in the case of N. gonorrhoeae (Fig. 2D). The exact location of the sialyltransferase is an outstanding question for most of these bacteria and it is our future aim to investigate the location of the sialyltransferases for the various bacteria used here, and whether the nucleotide sugars are taken up and metabolized intracellularly or transferred via ectopic glycosyltransferases from bacteria.

Fig. 4 A) Fluorescence microscopy images of N. meningitidis B fluorescently labeled using CMP-Neu5AF488 (488 channel). B) Fluorescence microscopy images of DAPI stain 405 channel C) merge of 488 and 405 (DAPI) channels.

We next investigated the results from the application of our method in more detail for the specific strains. From the genus Neisseria we selected a commensal strain, N. lactamica, and 12 pathogenic strains of N. meningitidis. N. lactamica is known to be present in the upper respiratory tract of humans (Dorey et al. 2019), and to confer natural immunity against meningitis in children. There are suggestions that the LOS could be implicated in this immunization (Gold et al. 1978; Kim et al. 1989; Maeland and Wedege 1989; Sánchez et al. 2002; Braun et al. 2004). Sialylation of N. lactamica LOS has been reported, and the presence of the lst gene has been detected in several N. lactamica strains, including the one used in our study DSM 4691. Tsang et al. (2001), also detected the lst gene in all the N. meningitidis LOS prototype strains (L1-L12) used in this paper. We confirmed these results by PCR (Supplementary information, Table S3, Fig. S9) using the primers designed by Tsang et al (Tsang et al. 2001).

MOE applied on N. lactamica and N. meningitidis L12, reveals the capability of these strains of metabolizing Neu5Az (Supporting information, Fig. S10). Our data shows that N. meningitidis strains L1-L10 and L12, and serotypes B, C, W-135 and Y, and the commensal N. lactamica, are all capable of utilizing CMP-Neu5Az to incorporate sialic acid into their LOS, similar to N. gonorrhoeae (Fig. 5A–C) (Jen et al. 2021). Strain L6 which lacks the terminal galactose residue could be sialylated, but to a lesser extent compared to other strains. This may indicate that the epitope found in the L6 strain LOS (4GlcNAcβ1–3Galβ1–4Glc) (Mubaiwa et al. 2017) is not the most suitable substrate for its own sialyltransferase. Sialylation of strain L12 is also reduced compared to the others, however since the LOS of this strain is not fully elucidated yet we cannot hypothesize about the acceptor preference of the ST. Furthermore, we do not have information about the level of expression of the Lst enzyme within the different strains. The lack of sialylation of the LOS of N. meningitidis L11 could also indicate the specificity of the enzyme for substrates containing Galβ1–4GlcNAcβ1–3Galβ1–4Glc, or to a lesser extent, 4GlcNAcβ1–3Galβ1–4Glc, but not for Glcβ1–4Glc which is the epitope found in the L11 LOS (Mistretta et al. 2010). In the literature there is structural evidence of the presence of Neu5Ac in the LOS of N. meningitidis strains L1-L5 (Mandrell et al. 1991; Choudhury et al. 2008), strains L9-L12 belong to the serogroup A (Tsang et al. 2001), this group does not have Neu5Ac in the CPS, and this explains why sialic acid has not been found in the LOS of strains cultured without an external supply of Neu5Ac.

Fig. 5 Scan for the sialyltransferase activity of N. meningitidis strains with CMP-Neu5Az. LOS was visualized on a silver-stained LOS/LPS gel, and sialylated LOS was detected in western blot via SPAAC reaction with DBCO-PEG4-biotin.

From the genus Campylobacter, we observed labeling of different strains of C. jejuni GBS strains, C. upsaliensis, and C. insulaenigreae. Among these, C. upsaliensis and C. insulaenigrae are considered emerging zoonotic pathogens for humans (Fouts et al. 2005; Chua et al. 2007; Man 2011; Richards et al. 2013). Little is known about the metabolism of Neu5Ac in these two bacteria, or the LOS structure that they express. Richards et al. (2013) reported the genomic potential for sialic acid biosynthesis of strains of C. upsaliensis and C. coli. To confirm the presence of these genes in our strain we designed primers targeting parts of the cstII gene based on the published C. jejuni sequence and found it is indeed present in C. upsaliensis and C. insulaenigreae. For the C. coli and C. lari strains in our lab, we did not detect the cstII genes, which is consistent with our LOS labeling results. Our labeling data showed that C. upsaliensis DMSZ 5365 and C. insulaenigrae DSM 17739 are indeed capable of transferring CMP-Neu5Ac onto their LOS, translating the genomic potential of these strains to functional evidence for a LOS active sialyltransferase and perhaps a role of sialylated LOS in its pathogenicity (Fig. 6A).

Fig. 6 Scan for native ST activity of (A) campylobacter strains and (B) haemophilus strains with CMP-Neu5Az. Detected on a silver-stained LOS/LPS western blot via SPAAC reaction with DBCO-PEG4-biotin.

For several Haemophilus species, it has been reported that their LOS can resemble human glycans (de Jong et al. 2022a). This form of glycan mimicry has also been observed for some strains of the pathogen H. aegyptius (Rubin 1995; Swords et al. 2003; Heise et al. 2018). Sialylation of H. aegyptius LOS has however not been described yet, but we here show H. aegyptius DSM 21187 expresses active STs capable of introducing Neu5Ac onto its LOS (Fig. 6B). This indicates it might perform a similar function in immune evasion as for non-typeable H. influenzae (Saha et al. 2021). For strains of commensal H. parainfluenzae, the LOS or LPS has been characterized and Neu5Ac was not reported to be present in these glycan structures (Pollard et al. 2008; Vitiazeva et al. 2011; Young and Hood 2013). One report had scanned for the genetic potential to express an α2,3-sialyltransferase (lic3A), but could not identify this for any of the commensal bacteria (Young and Hood 2013). Another report identified Neu5Ac as part of the O-antigen of strain 20 (Vitiazeva et al. 2011). Our results here indicate that H. parainfluenzae strain DSM 8978 expresses active STs that introduce Neu5Ac onto its LOS. We designed primers to assess the presence of genes for Lic3A, LsgB and SiaA, and consistent with previous findings, we were able to find it in H. aegyptius but not in H. parainfluenzae.

To test the practical applicability of our method to quickly type ST activity in clinical isolate collections, we screened 10 clinical non-typeable and 2 typeable isolates of H. influenzae using our protocol in combination with CMP-Neu5biotin. The LOS and sialylation ability of these non-typeable and typeable clinical isolates was unknown and we found that seven out of ten showed sialylation of their LOS, and a moderate signal was detected for both typeables strains (Fig. 7). With this example, we demonstrate that this technique provides a rapid way to explore the sialyltransferase activity of multiple strains and to identify the sialylation of previously uncharacterized cell surface glycoconjugates of clinical isolates. Moreover, this NTHi sialyltransferase activity screen is also a rapid method for identifying functional sialylation of LOS, which provides a quick indication of whether the bacteria may use exogenous CMP-Neu5Ac in vivo.

Fig. 7 The native sialyltransferases of non-typeable and typeable H. influenzae strains show incorporation of CMP-Neu5Biotin to varying degrees, as observed after a strain-promoted click reaction with a DBCO-PEG4-biotin.

We investigated the animal pathogen P. multocida serovar 3 strains Pm70 and P1059 that are known to display sialylated LOS/LPS (Harper et al. 2011), which is thought to shield the bacteria from the immune system (Tatum et al. 2009). P. multocida has a known sialyltransferase, Pmst, which also has sialidase activity (Yu et al. 2005). In our experiment, P. multocida DSM 5281 showed modest labeling of the LOS by native sialyltransferases (Fig. 8A). It is possible that the experimental conditions were not optimal for sialyltransferase activity, or that the sialidase activity of the enzyme also played a role. We tested another strain of the genus Pasteurella, P. dagmatis, an animal and human pathogen (Holst et al. 1992). This bacterium has a known sialyltransferase, PdST, which catalyzes the transfer of CMP-Neu5Ac onto the galactosyl residue of the LOS/LPS with an α2,3-linkage (Schmölzer et al. 2013). This enzyme hydrolyzes CMP-Neu5Ac in the absence of an acceptor substrate (Schmölzer et al. 2014). We performed MOE on this bacterium and observed labeling of the LOS (Supporting information, Fig. S10). In agreement with this result, the LOS/LPS of P. dagmatis was labeled via our method (Fig. 8A).

Fig. 8 Scan for the sialyltransferase activity of (A) Prevotella, and (C) Pasteurella strains. CMP-Neu5Az was incorporated by native sialyltransferases followed by a strain-promoted click reaction with a DBCO-PEG4-biotin. The LOS/LPS was analyzed on western blot and by silver stain.

Finally, we also studied two sialidase positive Prevotella strains P. timonensis 5C-1B and P. bivia both associated with bacterial vaginosis (Pelayo et al. 2024; Segui et al. 2024). Little is known for these bacteria about their utilization of Neu5Ac, and their potential to display glycan mimicry (Srinivasan et al. 2015). By applying MOE, we found that strains of P. timonensis 5C-B1, TSW and ACES are capable of metabolizing and incorporating Neu5Az onto their LOS, but not P. bivia (Supplementary information, Fig. S10). This suggests the presence of the enzymatic machinery capable of metabolizing Neu5Ac in the P. timonensis strains. Labeling experiments using CMP-Neu5Az also show that P. timonensis 5C-1B and P. bivia have active STs capable sialylating their LOS with exogenous CMP-Neu5Ac (Fig. 8B). To the best of our knowledge, sialyltransferases from P. timonensis have not been described in the literature or have not been annotated in the genome yet. Taken together, these data suggest that Neu5Ac can be used by this bacterium to decorate its glycoconjugates, and we were able to assess this easily using our NST glycoengineering approach.

Experimental section

Material, methods and culturing conditions

Neu5Az, CMP-Neu5Az, CMP-Neu5Biotin, and CMP-Neu5AF488 were prepared as reported (de Jong et al. 2022b). HRP conjugated anti-biotin antibody (200-032-211) was purchased from Jackson ImmunoResearch Laboratories. Acetylene-PEG4-biotin (CLK-TA105), DBCO-PEG4-biotin (CLK-A105P4), AF488-alkyne (CLK-1277), and DBCO-AF488 (CLK-1278) were purchased from Jena Bioscience. Normal Human Serum (NHS) was a kind gift from UMC Utrecht. Multicolor low range protein ladder (26628) and ProLong Diamond Antifade Mountant (P36961) from, DAPI (4′,6-diamidino-2-phenylindole) (Merk), ECL Western Blotting Substrates (Bio-Rad Laboratories), Alcian Blue 8GX (Fluka, Sigma-Aldrich), DNA extraction kits (Macherey-Nagel GmbH & Co.), protein ladder and 1 Kb DNA ladder (Thermo Fisher Scientific), primers were ordered from Macrogen Europe. Agar blood sheep plates purchased from BioTrading (K004P090KP), chocolate plates (BioTrading, K018P090KP), chopped meat media and NYC media (DSMZ), trypticase soy yeast extract Oxoid™ (Thermo Fisher Scientific), heart infusion broth (BioTrading, K716F100GH), HEPES (Merk). The sialyltransferases CstII, Pmst1, Pmst3, Pd2,3-ST were expressed and purified in house (Utrecht University). All bacteria strains cultured as specified in the Supporting information. Further information on the bacterial strains used in this work can be found in Supporting information, Table S1.

SEEL of bacteria: (de Jong et al. 2022b)

One-step SEEL was performed on bacteria grown in liquid culture (5 × 108 bacteria). The bacteria were washed with buffer and were incubated with SEEL label mix at 37 °C for 2 h while rotating. A typical SEEL label mix (50 μL) was prepared in medium (PBS/HEPES buffer) with sialyltransferase (1.05 uL of stock 1 mg/mL, or 2 mg/mL in case of Pmst3 and Pd2,3-ST), CMP-sialic acid derivative (50 μM), 0.34 μL BSA (2 mg/mL) and 0.34 μL alkaline phosphatase (1 U/μL). After SEEL treatment, the bacteria were washed with buffer and prepared for application. Two-step SEEL was performed similar to one-step SEEL, followed by a click reaction. In case of CuAAC, 100 μL reaction volume contained 100 μM acetylene-PEG4-biotin, 500 μM CuSO4 and 2.5 mM sodium L-ascorbate. In case of SPAAC 100 μL reaction volume contained 100 μM DBCO-PEG4-biotin. For the fluorophores AF488-alkyne and DBCO-AF488 the concentration was 1 mM. If the bacteria were heat-inactivated, they were heated at 80 °C for 15 min and treated with the described SEEL method.

MOE with Neu5Az

Neu5Az were synthesized according to a published procedure (Luchansky et al. 2004; Han et al. 2005). MOE was performed with 6 × 108 bacteria, and these were incubated with indicated concentrations of Neu5Az and Neu9Az. Bacteria were incubated for 6 h shaking at 160 rpm and for NTHi at 37 °C, and for C. jejuni at 42 °C under microaerophilic conditions. After incubation, the samples were washed 2 × 1 mL (buffer) and clicked via CuAAC or SPAAC, see SEEL of bacteria, washed again and further treated for (glyco)proteins or LOS.

Labeling of bacterial LOS via native sialyltransferases

Bacteria were grown as described previously. Bacteria (5 × 108 CFU/mL) were centrifuged at 5,000 rpm for 5 min and washed with HEPES (in case of Neisseria) or PBS. For two step labeling, bacterial pellet was resuspended in a solution of the sugar nucleotide (CMP-Neu5Az) in buffer at a final concentration of 50 μM, and incubated for 2 h at 37 °C on rotating device. For negative controls bacteria were incubated with buffer. Bacteria, including negative controls, were washed 2 times with buffer and incubated 1 h with a solution of DBCO-PEG4-biotin 100 μM or DBCO-AF488, at room temperature. After this, bacteria were washed multiple times and resuspended in PBS. For one step labeling, we incubated the bacteria with a solution 50 μM of CMP-Neu5biotin or CMP-Neu5AF488 for 2 h at room temperature. Bacteria were washed multiple times with PBS.

For LOS visualization samples were boiled for 5 min and then treated with proteinase K (10 μL, 20 mg/mL) overnight at 55 °C.Laemmli buffer was added and loaded onto tris-tricine gels as described below. Western blot using anti-biotin HRP was employed to detect labelled LOS when CMP-Neu5biotin was used or two steps labeling using CMP-Neu5Az followed by SPAAC with DBCO-PEG4-biotin was performed. Loading controls were stained with the silver staining standard protocol described below.

In case of (glyco)proteins, the samples were lysed and analyzed with a 10% SDS-PAGE gel for which the gel was run for 45–60 min at 150 V. Western blots to detect biotin tags and Page Blue to stain loading controls.

To check sialylation of capsules (N. meningitidis B, C, W-135 and Y, and C. jejuni GB11 and GB19) 1 × 108 bacteria was centrifuged at 13,000 rpm for 5 min and resuspended in 60 μL of lysis buffer (0.32 mL 1 M Tris/HCl pH 6.8, 0.4 g SDS, 2.5 mg bromophenol blue, 2.0 mL glycerol, and 7.68 mL H2O). The supernatant was treated with 1.5 μL of a 20 mg/mL stock proteinase K, and heated at 50 °C for 1 h, followed by boiling at 100 °C for 10 min. Samples were loaded on 10% SDS-Page gel, and ran at 125 V. Western blot was used to detect biotinylated CPS. For the loading controls, an adapted protocol of Lin was applied (Boleij et al. 2018). Briefly, after electrophoresis, gels were washed in a solution 25% ethanol and 10% acetic acid for 3.5 h. Gels were stained in 0.125% Alcian Blue in 25% ethanol and 10% acetic acid (pH 2.5) for 1 h and followed by several washings with 25% ethanol and 10% acetic acid solution overnight, until bands appear.

Western blotting

For the western blotting, the gel was electroblotted onto a PVDF membrane. The membrane was blocked with 5% skimmed milk for 30–60 min, washed with 1% milk for 5 min, stained with anti-biotin-HRP antibody (1:20000 in 1% milk), washed (1% milk, followed by PBS, 5 min each), and treated with ECL Western substrate for signal detection.

LOS preparation and tris-tricine gel

Samples were loaded onto a 16% Tris-Tricine gel. The gel ran typically for 3–4 h at 20 mA and was then further analyzed by Western blotting and silver staining or in-gel fluorescence. The molecular weights of the LOS were not determined; however, some samples were subjected to electrophoresis alongside an E. coli LPS standard for comparative purposes.

Silver staining

Silver staining was performed on a 16% Tris-Tricine gel as described previously (Merril and Shifrin 1982). Briefly, the gel was fixed (30 min, 40% ethanol, 5% acetic acid), oxidized (5 min, 0.7% sodium periodic acid, 40% ethanol, 5% acetic acid), washed (3 × 5 min in distilled water), stained (distilled water containing 19% 0.1 M NaOH, 1.3% >28% ammonium hydroxide, 3.3% 20% w/v silver nitrate), washed (3 × 5 min in distilled water), developed until bands appeared (distilled water containing 0.1% PFA 37% and 0.1% citric acid 100 mg/mL), rinsed with distilled water and stopped (7% acetic acid in distilled water).

In-gel fluorescence

In-gel fluorescence was measured on Amersham imager 600 using the Green channel (520 nm, Cy3).

Serum resistance assay

N. gonorrhoeae WT was incubated with and without 20 nmol/mL CMP-Neu5Ac or CMP-Neu5Az. After treatment, N. gonorrhoeae was washed and diluted to 106 bacteria in HEPES buffer. Bacteria (1 × 104) were treated with 10% NHS or HI-NHS for 1 h at 37 °C. Samples were 25× diluted and 50 μL was plated out on chocolate columbia agar with vitox. Plates were grown at 37 °C + 5% CO2 overnight, colony forming units were counted and data analysis was done with Prism software.

Detection of genes by PCR

Bacteria were cultured as described previously. To extract chromosomal DNA, 1 mL of culture with an OD600 of 1 was centrifuged at 5,000 rpm for 5 min. The DNA was extracted from the pellet using the instructions provided by a commercial kit (Macherey-Nagel GmbH & Co.). Each reaction consisted of 500 nM of primer FWD and REV (Table S3, Supporting information), 200 nM of dNTP (Thermo Fisher Scientific), 5 U of DreamTaq DNA Polymerase (Thermo Fisher Scientific), and 1 μL of extracted DNA (15–25 μg/mL), in a total volume of 50 μL. PCR reactions were performed in a thermal cycler (MyCycler Bio-Rad) using a standard protocol. The cycling conditions consisted of an initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at the appropriate temperature for 30 s, and extension at 72 °C for 45 s, with a final extension period of 5 min. The amplification products were visualized by electrophoresis on a 1% agarose gel and Midori green staining, and then detected using a ChemiDoc MP system (Bio-rad). PCR products were sent to sequence without purification.

Fluorescence confocal microscopy

Bacteria (circa 3 × 108 cells) were centrifuged, resuspended in HEPES +1% BSA and DAPI (4′,6-diamidino-2-phenylindole) (1:50) was added. The samples were incubated for 25 min in the dark before washing (milliQ +0.1% Tween) and then carefully resuspended in ProLong Diamond Antifade Mountant (P36961). 10 μL of sample was taken, put on a poly-L-lysine coated coverslip and mounted on a glass slide. Slides were stored at RT overnight to allow the samples to harden. Images were collected on Olympus/Evident SpinSR10 confocal microscope in combination with SORA software. Image analysis was performed using OliVia software.

Conclusion

We demonstrate that labeling bacterial lipooligosaccharides (LOS) through native sialyltransferases (NSTs), MOE, and SEEL serve as a complementary glycoengineering techniques for studying the role of Neu5Ac in bacteria, with the choice among these methods depending on specific objectives. Using an expansive and diverse set of bacteria, many of which are relevant to human health and disease, we show that glycoengineering with NSTs, in particular, offers a straightforward, rapid, and non-toxic approach for adding various modifications to the bacterial surface. This NST labeling protocol also allows for quick and easy screening for the presence of active bacterial sialyltransferases and their potential to sialylate LOS with exogenous CMP-Neu5Ac, regardless of their ability to produce and metabolize Neu5Ac. This enabled us to annotate and validate, for the first time, that a wide array of bacterial pathogens exhibit LOS sialyltransferase activity, paving the way for more in-depth exploration of glycosylation processes in bacterial LOS. Further development of glycoengineering with NSTs will hopefully allow for precise labeling, visualization, and tracking of Neu5Ac on bacterial surfaces in the future. Another future direction of this research will focus on determining the exact localization of NSTs within bacterial cells, but the rapid labeling time observed using CMP-Neu5Az suggests the presence of NSTs near or on the bacterial outer surface, as demonstrated with N. gonorrhoeae. Finally, by harnessing a broad range of CMP-Neu5Ac analogs we will use this method to search for novel NSTs with broad donor specificity, which will find use in the field of chemoenzymatic synthesis and expand the toolkit for bacterial glycoengineering.

Supplementary Material

Supporting_information_NST_glycoengineering_FINAL_cwae071

Acknowledgments

We thank Prof. dr. Geert-Jan Boons and dr. Gerlof P. Bosman for providing the recombinant sialyltransferases used in the SEEL experiments. Prof. dr. Nina van Sorge and the Netherlands Reference Laboratory for Bacterial Meningitis (NRLBM) for generously providing the N. meningitidis strains L1-L12. Dr. Karin Strijbis and Celia Segui-Perez for providing the Prevotella strains described in this study. Nontypeable H. influenzae R2886 and the siaP mutant were a kind gift from dr. Jeroen Langereis, Radboudumc. Other nontypeable H. influenzae and typeable strains were a kind gift from Clinical Infectiology, Utrecht University. Normal Human Serum (NHS) was a kind gift from prof. dr. Suzan Rooijakkers from UMC Utrecht. Dr. Astrid Heikema from Erasmus MC Rotterdam for providing C. jejuni GB strains and their cstII mutants. Prof. dr. Jos van Putten for the N. gonorrhoeae and N. meningitidis serogroup B, C, W-135 and Y strains. Dr. Maria J. Moure for the Sia-AF488-CMP used in this study.

Author contributions

Erianna I. Alvarado-Melendez (Conceptualization, Formal analysis [equal], Investigation, Methodology, Resources, Validation, Writing—original draft, Writing—review & editing), Hanna de Jong (Conceptualization, Formal analysis, Investigation, Methodology, Resources, Validation, Writing—review & editing), Jet Hartman (Investigation), Jun Yang Ong (Resources), Marc Wösten (Conceptualization, Formal analysis, Supervision, Writing—review & editing), and Tom Wennekes (Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing—original draft, Writing—review & editing).

Funding

This work was supported by the Dutch Research Council (NWO) via a VIDI grant (723.014.005 to TW) and BBOL grant (737.016.013). Funding from the European Union’s Horizon 2020 Marie Skłodowska-Curie Actions for the Innovative Training Network “Sweet Crosstalk” under the grant agreement No 814102.

Conflict of interest statement

The authors declare no conflict of interest.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.
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