Multi-drug resistant strains of Acinetobacter baumannii are increasingly being isolated in hospitals worldwide. Among the virulence factors identified in this bacterium there is a general O-glycosylation system that appears to be important for biofilm formation and virulence, and the capsular polysaccharide, which is essential for resistance to complement killing. In this work, we identified a locus that is responsible for the synthesis of the O-pentasaccharide found on the glycoproteins. Besides the enzymes required for the assembly of the glycan, additional proteins typically involved in polymerization and transport of capsule were identified within or adjacently to the locus. Mutagenesis of PglC, the initiating glycosyltransferase prevented the synthesis of both glycoproteins and capsule, resulting in abnormal biofilm structures and attenuated virulence in mice. These results, together with the structural analysis of A. baumannii 17978 capsular polysaccharide via NMR, demonstrated that the pentasaccharides that decorate the glycoproteins are also the building blocks for capsule biosynthesis. Two linked subunits, but not longer glycan chains, were detected on proteins via MS. The discovery of a bifurcated pathway for O-glycosylation and capsule synthesis not only provides insight into the biology of A. baumannii but also identifies potential novel candidates for intervention against this emerging pathogen.

Acinetobacter baumannii, capsular polysaccharide, capsule biosynthesis, genetic locus, O-pentasaccharide

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,6,4,2	Ac
3,6,4,3	Ac
3,6,4,4	Ac
3,6,4	bDGlcpN3NA	102.5	54.5	55.8	72.0	75.2	?
3,6,6,2	Ac
3,6,6	bDGlcpN	103.0	56.7	75.2	71.0	76.9	61.9
3,6	aDGalp	99.2	68.5	81.7	78.1	70.6	72.3
3	bDGlcp	106.1	74.1	76.8	70.0	75.4	65.8
2	Ac
	bDGalpN	104.9	52.6	81.7	69.1	75.9	62.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,6,4,2	Ac
3,6,4,3	Ac
3,6,4,4	Ac
3,6,4	bDGlcpN3NA	5.09	3.83	4.03	4.97	4.06	-
3,6,6,2	Ac
3,6,6	bDGlcpN	4.48	3.69	3.54	3.46	3.45	3.78-3.93
3,6	aDGalp	4.93	3.72	3.90	4.35	4.05	3.72-4.07
3	bDGlcp	4.57	3.32	3.49	3.61	3.61	3.65-3.99
2	Ac
	bDGalpN	4.62	4.15	3.87	4.16	3.71	3.84


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,6,4,2	Ac
3,6,4,3	Ac
3,6,4,4	Ac
3,6,4	bDGlcpN3NA	102.5/5.09	54.5/3.83	55.8/4.03	72.0/4.97	75.2/4.06	
3,6,6,2	Ac
3,6,6	bDGlcpN	103.0/4.48	56.7/3.69	75.2/3.54	71.0/3.46	76.9/3.45	61.9/3.78-3.93
3,6	aDGalp	99.2/4.93	68.5/3.72	81.7/3.90	78.1/4.35	70.6/4.05	72.3/3.72-4.07
3	bDGlcp	106.1/4.57	74.1/3.32	76.8/3.49	70.0/3.61	75.4/3.61	65.8/3.65-3.99
2	Ac
	bDGalpN	104.9/4.62	52.6/4.15	81.7/3.87	69.1/4.16	75.9/3.71	62.4/3.84

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@@H](O)[C@@H](OC[C@H]2O[C@@H](O[C@@H]3[C@@H](NC(C)=O)[C@H](*)O[C@H](CO)[C@@H]3O)[C@H](O)[C@@H](O)[C@@H]2O)O[C@H](CO[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2NC(C)=O)[C@@H]1O[C@@H]1O[C@H](C(=O)O)[C@@H](OC(C)=O)[C@H](NC(C)=O)[C@H]1NC(C)=O

1030.937 g/mol (C₄₀H₆₂R₂N₄O₂₇, R = next and previous repeats, not counted in mol weight)