Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3-O-methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

Lipopolysaccharide, Pseudomonas aeruginosa, monoclonal antibody, glycoconjugate vaccine, common polysaccharide antigen, methylrhamnan

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,4,4,2,4,3	Me	57.5
4,4,4,2,4	aDRhap	102.5	67.6	81.9	79.3	69.0	18.0
4,4,4,2,4,4,3	Me	57.5
4,4,4,2,4,4	aDRhap	102.5	67.5	80.8	72.0	69.1	18.0
4,4,4,2,3	Me	57.5
4,4,4,2	aDManp	102.5	67.4	81.8	73.9	73.3	62.2
4,4,4	aDManp
4,4	aDManp	102.6	71.9	72.1	75.4	73.5	62.2
4	aDManp	102.5	71.9	72.1	75.4	73.5	62.2
2	Me	59.7
	aDManp	99.4	83.1	71.2	75.6	?	?


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,4,4,2,4,3	Me	3.46
4,4,4,2,4	aDRhap	5.13	4.20	3.55	3.58	3.86	1.33
4,4,4,2,4,4,3	Me	3.46
4,4,4,2,4,4	aDRhap	5.15	4.22	3.42	3.50	3.82	1.29
4,4,4,2,3	Me	3.46
4,4,4,2	aDManp	5.27	4.28	3.62	3.85	3.78	3.78-3.86
4,4,4	aDManp	5.23	4.03	3.94	3.85	3.74	3.78-3.86
4,4	aDManp	5.21	4.01	3.94	3.85	3.74	3.78-3.86
4	aDManp
2	Me	3.47
	aDManp	5.37	3.66	3.96	3.76	?	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,4,4,2,4,3	Me	57.5/3.46
4,4,4,2,4	aDRhap	102.5/5.13	67.6/4.20	81.9/3.55	79.3/3.58	69.0/3.86	18.0/1.33
4,4,4,2,4,4,3	Me	57.5/3.46
4,4,4,2,4,4	aDRhap	102.5/5.15	67.5/4.22	80.8/3.42	72.0/3.50	69.1/3.82	18.0/1.29
4,4,4,2,3	Me	57.5/3.46
4,4,4,2	aDManp	102.5/5.27	67.4/4.28	81.8/3.62	73.9/3.85	73.3/3.78	62.2/3.78-3.86
4,4,4	aDManp	NMR TSV error 2: unequal length of 13C and 1H datasets
4,4	aDManp	102.6/5.21	71.9/4.01	72.1/3.94	75.4/3.85	73.5/3.74	62.2/3.78-3.86
4	aDManp	NMR TSV error 2: unequal length of 13C and 1H datasets
2	Me	59.7/3.47
	aDManp	99.4/5.37	83.1/3.66	71.2/3.96	75.6/3.76	?/?	?/?

The spectrum also has 2 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CO[C@@H]1[C@@H](O)O[C@H](CO)[C@@H](O[C@H]2O[C@H](CO)[C@@H](O[C@H]3O[C@H](CO)[C@@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O[C@H]5O[C@H](C)[C@@H](O[C@H]6O[C@H](C)[C@@H](O[C@H]7O[C@H](C)[C@@H](O[C@H]8O[C@H](C)[C@@H](O[C@H]9O[C@H](C)[C@@H](O)[C@H](OC)[C@@H]9O)[C@H](OC)[C@@H]8O)[C@H](OC)[C@@H]7O)[C@H](OC)[C@@H]6O)[C@H](OC)[C@@H]5O)[C@H](OC)[C@@H]4O)[C@H](O)[C@@H]3O)[C@H](O)[C@@H]2O)[C@@H]1O

1657.619 g/mol (C₆₇H₁₁₆O₄₆)