Pseudaminic acid on exopolysaccharide of Acinetobacter baumannii

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Pseudaminic acid on exopolysaccharide of Acinetobacter baumannii plays a critical role in phage-assisted preparation of glycoconjugate vaccine with high antigenicity. I-Ming Lee, Feng-Ling Yang, Te-Li Chen, Kuo-Shiang Liao, Chien-Tai Ren, Nien-Tsung Lin, Yu-Pei Chang, Chung-Yi Wu, and Shih-Hsiung Wu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04078 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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Journal of the American Chemical Society

Pseudaminic acid on exopolysaccharide of Acinetobacter baumannii plays a critical role in phage-assisted preparation of glycoconjugate vaccine with high antigenicity. I-Ming Lee†, Feng-Ling Yang†, Te-Li Chen , Kuo-Shiang Liao‡, Chien-Tai Ren‡, Nien-Tsung Lin§, Yu-Pei Chang†, Chung-Yi Wu*,‡ and Shih-Hsiung Wu*,†,#,⊗ ∥

†

Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

‡

Genomics Research Center, Academia Sinica, Taipei 115, Taiwan

§

Master program in Microbiology and Immunology, Tzu Chi University, Hualien 970, Taiwan

∥

#

Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei 112, Taiwan

Department of Chemistry, National Taiwan University, Taipei 106, Taiwan

⊗

Institute of Biochemical Science, National Taiwan University, Taipei 106, Taiwan

Supporting Information Placeholder ABSTRACT: Pseudaminic acid (Pse) has been known for participating in crucial bacterial virulence and thus is an attractive target in the development of glycoconjugate vaccine. Particularly, this therapeutic alternative was suggested to be a potential solution against antibiotic resistant Acinetobacter baumannii that poses a serious global health threat. Also, Pse was found to be involved in the exopolysaccharide (EPS) of mild antibiotic resistant A. baumannii strain 54149 (Ab-54149) of which specific glycosyl linkage can be depolymerized by phageΦAB6 tailspike protein (ΦAB6TSP). In this study, we found that the antibodies induced by Ab-54149 EPS was capable of recognizing a range of EPS of other clinical A. baumannii strains, and deemed as a great potential material for vaccination. To efficiently acquire homogeneous EPS-derived oligosaccharide with significant immunogenic activity for the production of glycoconjugate, we used the ΦAB6TSP for the fragmentation of Ab-54149 EPS instead of chemical methods. Moreover, insight into the ligand binding characterization of ΦAB6TSP suggested the branched Pse on the Ab-54149 EPS served as a recognition site of ΦAB6TSP. The serum boosted by ΦAB6TSP-digested product and carrier protein CRM197 conjugate complex displayed specific sensitivity toward Ab-54149 EPS with bacterial killing activity. Strikingly, Pse is an ideal epitope with strong antigenicity, profiting the application of the probe for pathogen detection and glyco-based vaccine. Pseudaminic acid (Pse) belongs to the family of sugars called nonulosonic acid and is found as a component unique to surface glycan and glycoproteins of bacterial pathogens. Pse was first discovered from lipopolysaccharide of Pseudomonas aeruginosa O7/O9 and has been considered to be an important virulence factor in pathological process.1,2 Notably, the post translational Pse modification of flagellin subunits is necessary for functional flagellin assembly in C. jejuni and H. pylori to facilitate colonization.3,4 Most of all, Pse may contribute to bacterial virulence by dampening host immune response due to the structural similarity to eukaryote sialic acid.5-8 Accordingly, Pse has drawn tremendous attention to be an attractive target for new therapeutic application over recent years, although the exact biological function of Pse remains elusive. Pse has been found on the exopolysaccharide of numerous Gram-negative pathogens, including Acinetobacter baumannii (A.

baumannii). A. baumannii is one of the most serious opportunistic pathogens responsible for nosocomial infection in the modern by causing diseases such healthcare system9-11 as ventilator-associated pneumonia, bloodstream infections, and meningitis with high mortality.12-14 During the past decade, antibiotic resistant A. baumannii has become increasingly alarming.13,15 The “last-line” antibiotics including carbapenem and colistin for the management of Gram-negative pathogen prevalence are getting less effective.16-20 Hence, a new therapy is urgently required to overcome the failure of antibiotic treatment. Glycoconjugate vaccines have been proven to be effective in combating a broad spectrum of diseases, particularly the infectious diseases caused by antibiotic resistant bacteria.21-23 In this regard, selection of targeted microbial exopolysaccharide (EPS) that elicits strong immune response with extensive strains coverage is prerequisite for glycoconjugate preparation.24,25 Furthermore, some earlier studies demonstrated that glycoconjugate with proper repeat units of EPS exhibited better bactericidal activity compared to those with whole EPS.24,26-29 However, using traditional chemical hydrolysis of EPS generated heterogeneous oligosaccharide fragments, which impede the reproducibility of glycoconjugate.30 Although, chemical synthesis of designed oligosaccharide could produce homogeneous oligosaccharides but was time consuming and had low yield.31 Utilizing phage tailspike protein (TSP) that is capable of hydrolyzing the specific glycosyl linkage on EPS posed a possible solution.32,33 Therefore, insight into the substrate recognition of TSP and the immunogenicity of TSP-hydrolyzed products will advance the preparation of glycoconjugate vaccine. The A. baumannii strain 54149 (Ab-54149) is a mild antibiotic resistant clinical strain isolated from patients in the intensive care unit (ICU). Based on our prior report, the structure of A. baumannii strain 54149 (Ab-54149) EPS was comprised of a repeat unit of →3)-β-GalNAcp-(1→3)-[β-Glcp-(1→6)]-βGalp-(1→ and the majority of the repeat unit contained a pseudaminic acid (Pse) connected to Glcp via a Pse-(2→6)-α-Glcp linkage, sharing the same structure with other aminoglycoside and carbapenem resistant A. baumannii strains34-36 (GalNAcp: N-acetyl galactosamine, Galp: galactose, Glcp: glucose) (Fig. S1). Additionally, we verified that the phage ΦAB6 tailspike protein (ΦAB6TSP) can depolymerize Ab-54149 EPS and consequently generate two repeat units as the major product

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(compound 1). With respect to the ΦAB6TSP-Ab-54149 EPS complex crystal structure, the Pse was found to be embedded stably in surface cavity of ΦAB6TSP with three hydrogen bonds, implying that Pse was implicated in substrate recognition of ΦAB6TSP.36 In the present study, Ab-54149 EPS was used as antigen and injected into mice. Ab-54149 EPS-induced antibodies could recognize Ab-54149 EPS, compound 1 and approximately 40% of EPS extracted from other 250 A. baumannii clinical strains (Fig. S2), suggesting Ab-54149 EPS is a valuable target for development of glycoconjugate vaccine. For preparation of Ab-54149 EPS-derived oligosaccharide glycoconjugate, ΦAB6TSP was believed to be as a useful tool.37 To understand the substrate-binding model of ΦAB6TSP, we monitored the quantitative evaluation of saturation transfer difference (STD) NMR effects of Ab-54149 EPS when it is bound to ΦAB6TSP in solution. The STD NMR spectrum revealed apparent signal main for the Pse on Ab-54149 EPS; the highest levels of saturation were corresponding to the protons on C-7, C-9 and C-3, and inferior levels of saturation were corresponding to the protons on C-6, C-8 of Pse (Fig. 1A and 1B). Owing to close connection with Pse, the protons on C-2, C-3, C-4 and C-6 of Glcp experienced STD effect as well (Fig. 1B). This EPS-binding model depicted by STD NMR was in agreement with the complex structure model (Fig. 1C).

Ab-54149 EPSa demonstrated near identical elute pattern, suggesting that the Ab-54149 EPS and Ab-54149 EPSa possessed similar molecular weights (Fig. S6A). Besides, two Pse were absent by manipulating the compound 1 under above acidic condition (Fig. S5A and S5B). These results elucidated that Ab-54149 EPSa substantially removed partial Pse without altering the number of repeat units compared to Ab-54149 EPS. Then, the enzymatic activity and kinetics of ΦAB6TSP toward Ab-54149 EPS and Ab-54149 EPSa were quantified by 3,5-dinitrosalicylic acid (DNS) assay.39 We noticed that of ΦAB6TSP reduced the ability of Ab-54149 EPSa digestion in a time dependent manner, as distinct from Ab-54149 EPS (Fig. S6B). This phenomenon can be interpreted by kinetic assay in which the absence of Pse on EPS dramatically reduces the Michaelis constant (Km) of ΦAB6TSP with a slight change on turnover rate (kcat) (Table 1, Fig. S7A and S7B). The variation of Km was accounted for the binding affinity of enzyme toward the substrate. Indeed, microscale thermophoresis (MST) measurements showed that the affinity constant (KD) of Ab-54149 EPS to ΦAB6TSP was almost seventy-fold higher than that of Ab-54149 EPSa (Fig. 2). Thus, we provided the compelling evidences verifying that Pse was the critical recognition site for ΦAB6TSP substrate binding.

Figure 1. STD NMR analysis of Ab-54149 EPS bound to ΦAB6TSP. (A) 1H NMR spectrum of Ab-54149 EPS (color in red) and STD NMR spectrum (color in blue) of Ab-54149 EPS in the presence of ΦAB6TSP: ligand ratio equals to 1: 50. (B) STD-derived epitope mapping of ΦAB6TSP: Ab-54149 EPS interaction with color coding from highest (red) to lowest (yellow) observed STD effect. (C) The ΦAB6TSP substrate complex structure: hydrogens on Glcp (colored in yellow) and Pse (colored in light purple) in the proximity of Pse binding site. To investigate the role of Pse toward ΦAB6TSP, we characterized the kinetic and binding affinity of ΦAB6TSP when reacted with Ab-54149 EPS that lacked of Pse. A study indicated that nonulosonic acid on polysaccharide, especially for sialic acid, could be released and purified by using mild acetic acid hydrolysis.38 Since the structure of Pse is similar to sialic acid, we treated Ab-54149 EPS with acetic acid to form Ab-54149 EPSa followed by dialysis with membrane cutoff 100~500 Da. Water outside the dialysis membrane was collected to lyophilize and then analyzed by ESI-Mass. Molecular weight of major component in water was identical to Pse (335 Da) and its isotope mass pattern is consistent with anticipated isotope mass spectrum of Pse (Fig. S3A and S3B). Although 1H NMR spectra showed that stoichiometry of Pse in Ab-54149 EPSa was much smaller in contrast to Ab-54149 EPS (Fig. S4A, S4B and S4C), size exclusive HW-65F column analysis of Ab-54149 EPS and

Figure 2. Microscale thermophoresis (MST) binding measurement of ΦAB6TSP toward Ab-54149 EPS (A) and Ab-54149 EPSa (B). Data are mean ± standard deviation from three independent experiments.

Table 1. Enzymatic hydrolysis of kinetic parameters of ΦAB6TSP toward Ab-54149 EPS and Ab-54149 EPSa


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Scheme 1. ΦAB6TSP digested product (compound 1) covalently conjugated to carrier protein CRM197. Pse is colored in pink.

Subsequently, the thiol-maleimide coupling method was adopted to produce glycoconjugate using compound 1 as target40 (Scheme 1). Amine derived compound 1 was reacted with commercially available amine reactive reagent 3,3-dithiobis(sulfosuccinimidylpropionate) (DTSSP) in PBS buffer (pH 7.4) overnight. Next, the disulfide bond was cleaved with dithiothreitol (DTT) at 40 oC for 1 hour to obtain the thiol products 1. On the other hand, carrier protein diphtheria toxin (DT) mutant CRM197 was reacted with N-(ε-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS) in PBS (pH 8.0) for 1 hour to form maleimide group on the protein. Eventually, glycoconjugate 3 was obtained by mixing thiol oligosaccharide 2 and maleimide-modified CRM197 in PBS (pH 7.2) for 1 hour. The number of maleimide linkers and glycoconjugated 3 on the CRM197 were determined by MALDI-TOF mass spectrometry. The results showed we successfully coupled close to 21 maleimide linkers to one CRM197 molecule, and 4 of 21 maleimide linkers were modified by compound 1 (Fig. S8).

monosaccharide that was released from Ab-54149 EPS via mild acetic acid hydrolysis over time rather than other components on Ab-54149 EPS, including sialic acid, GalNAcp, Galp and Glcp (Fig 3D). Presumably, the specificity of antibodies recognition was attributed to the major structural differences between Pse and sialic acid of the N-acetyl group on C7 and the hydroxyl group on C9 with opposite chirality of C5, C7 and C8.

This glycoconjugate 3 was used to immunize rabbits with four times boosting in two weeks interval, and sera were collected for dot blot analysis. Herein, boosted sera from the rabbits could recognize both Ab-54149 EPS and compound 1, indicating an excellent immunogenicity of the compound 1 (Fig. S9A). Notably, boosted serum is sensitive toward Ab-54149 EPS than compound 1. Moreover, the dot blot results suggested that Ab-54149 EPS-induced and glycoconjugate 3 boosted antibodies aimed at similar antigen because both antibodies were able to recognize Ab-54149 EPS and compound 1. Hence, we speculated that glycoconjugate 3 boosted sera could exhibit broad coverage with EPS from other 250 A. baumannii clinical strains like Ab-54149 EPS-induced antibodies. However, the boosted sera exhibited weak cross-reaction on different EPS from other A. baumannii strains and bacteria, suggesting high specificity on Ab-54149 EPS (Fig. S9B). As expected, boosted sera displayed excellent binding capacity against Ab-54149 (Fig. S10) and led to apparent killing of live Ab-54149 via bactericidal assay (Fig. 3A). Remarkably, although Ab-SK44 EPS and Ab-54149 EPS shared most sugar components except for Pse41, the almost none recognition on Ab-SK44 EPS by serum implied that Pse played a critical role in antigenicity. The boosted sera failed in recognition while the Pse on Ab-54149 EPS and ΦAB6TSP-digested product was diminished, confirming the importance of Pse (Fig. 3C). Most strikingly, boosted serum particularly bound to Pse



ASSOCIATED CONTENT

Supporting Information Experimental material, methods and supplementary figures 1-7 can be found in the supporting information is available free of charge on the Webpage: http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *[email protected]. *[email protected]

Present Addresses Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by grants from Ministry of Science and Technology (grants No. 106-0210-01-15-02 and 104-0210-01-09-02) and Institute of Biological Chemistry, Academia Sinica. We acknowledge the use of instrument in the Biophysics Core Facility, Scientific Instrument Center at Academia Sinica. We also thank the staffs in the Mass Core and NMR core in the Genomic Research Center, Academia Sinica for assistance in ESI-MS, MALDI-TOF-MS and STD NMR performance.

REFERENCES

Figure 3. Bactericidal test and dot blot assay of glycoconjugate 3 boosted serum. (A) Complement bactericidal activity in serial dilutions (3x to 729x) of boosted serum. Percentage of bacterial death is evaluated by counting CFU on each agar plate (the insert). Data are mean ± standard deviation from three independent experiments. (B) 3000x diluted serum reacted with various amount of Ab-54149 EPS, de-Pse Ab-54149 EPS, compound 1 and de-Pse compound 1, respectively. (C) On the left panel, 3000x diluted serum react with Pse releasing from Ab-54149 EPS over time (acetic acid hydrolysis for 40, 80 and 120 minutes) and Ab-54149 EPS. Pse from hydrolysis for 120 minutes and Ab-54149 EPS were quantified to 10 µg. Concentration of a line was 10 times higher than b line. On the right panel, 3000x diluted serum was treated with 10 µg and 1 µg sialic acid, GalNAcp, Galp and Glcp, respectively. In this study, we demonstrated that Ab-54149 EPS was an efficacious target for development of glycoconjugate vaccine against antibiotic resistant A. baumannii. Hence, we further accomplished the facile phage-assisted preparation of Ab-54149 EPS-derived glycoconjugate via ΦAB6TSP and confirmed that glycoconjugate boosted serum actually kills live bacteria in vitro. Meanwhile, we discovered that Pse plays a critical role in fragmentation of Ab-54149 EPS by ΦAB6TSP and recognition of glycoconjugate-boosted serum. Pse represented the strong antigenicity like legionaminic acid, also a nonulosonic acid from bacterial EPS.42,43 These efforts provide a highly potent prophylaxis toward Pse-coated pathogen that may escape from host immune system and demonstrate that Pse is a new attractive glycotope for vaccination or immunotherapy.

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Pseudaminic acid on exopolysaccharide of Acinetobacter baumannii

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