A comprehensive understanding of capsular polysaccharide (CPS) diversity is critical to implementation of phage therapy to treat panresistant Acinetobacter baumannii infections. Predictions from genome sequences can assist identification of the CPS type but can be complicated if genes outside the K locus (CPS biosynthesis gene cluster) are involved. Here, the CPS produced by A. baumannii clinical isolate 36-1454 carrying a novel K locus, KL127, was determined and compared to other CPSs. KL127 differs from KL128 in only two of the glycosyltransferase (gtr) genes. The K127 unit in 36-1454 CPS was the pentasaccharide β-D-Glcp-(1→6)-D-β-GalpNAc-(1→6)-α-D-Galp-(1→6)-β-D-Glсp-(1→3)-β-D-GalpNAc in which D-Glcp at position 4 replaces d-Galp in K128, and the glycosyltransferases encoded by the different gtr genes form the surrounding linkages. However, although the KL127 and KL128 gene clusters encode nearly identical Wzy polymerases, the linkages between K units that form the CPS chains are different, i.e., β-D-GalpNAc-(1→3)-D-Galp in 36-1454 (K127) and β-D-GalpNAc-(1→4)-D-Galp in KZ-1093 (K128). The linkage between K127 units in 36-1454 is the same as the K-unit linkage in five known CPS structures, and a gene encoding a Wzy protein related to the Wzy of the corresponding K loci was found encoded in a prophage genome in the 36-1454 chromosome. Closely related Wzy proteins were encoded in unrelated phage in available KL127-carrying genomes. However, a clinical isolate, KZ-1257, carrying KL127 but not the prophage was found, and K127 units in the KZ-1257 CPS were β-D-GalpNAc-(1→4)-D-Galp linked, confirming that WzyKL127 forms this linkage and thus that the phage-encoded WzyPh1 forms the β-D-GalpNAc-(1→3)-D-Galp linkage in 36-1454. IMPORTANCE Bacteriophage therapy is an attractive innovative treatment for infections caused by extensively drug resistant Acinetobacter baumannii, for which there are few effective antibiotic treatments remaining. Capsular polysaccharide (CPS) is a primary receptor for many lytic bacteriophages, and thus knowledge of the chemical structures of CPS produced by the species will underpin the identification of suitable phages for therapeutic cocktails. However, recent research has shown that some isolates carry additional genes outside of the CPS biosynthesis K locus, which can modify the CPS structure. These changes can subsequently alter phage receptor sites and may be a method utilized for natural phage resistance. Hence, it is critical to understand the genetics that drive CPS synthesis and the extent to which genes outside of the K locus can affect the CPS structure.

Acinetobacter baumannii, capsular polysaccharide, phage, Wzy polymerase, K locus, K127

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,6,6,6	bDGlcp	104.0	74.3	76.9	70.9	77.2	62.0
3,6,6,2	Ac	175.7-176.3	23.7-24.1
3,6,6	bDGalpN	103.2	53.6	72.2	69.2	75.0	70.3
3,6	aDGalp	99.5	68.7	80.4	70.4	70.4	70.9
3	bDGlcp	105.8	74.2	76.9	70.2	75.5	66.0
2	Ac	175.7-176.3	23.7-24.1
	bDGalpN	104.1	52.7	81.4	69.1	75.8	62.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,6,6,6	bDGlcp	4.53	3.31	3.50	3.39	3.47	3.73-3.93
3,6,6,2	Ac	-	2.03-2.06
3,6,6	bDGalpN	4.50	3.89	3.73	3.97	3.87	3.94-4.07
3,6	aDGalp	4.96	3.90	3.91	4.19	4.04	3.72-4.05
3	bDGlcp	4.56	3.32	3.52	3.61	3.61	3.69-4.00
2	Ac	-	2.03-2.06
	bDGalpN	4.71	4.06	3.89	4.15	3.69	3.77-3.80


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,6,6,6	bDGlcp	104.0/4.53	74.3/3.31	76.9/3.50	70.9/3.39	77.2/3.47	62.0/3.73-3.93
3,6,6,2	Ac		23.7-24.1/2.03-2.06
3,6,6	bDGalpN	103.2/4.50	53.6/3.89	72.2/3.73	69.2/3.97	75.0/3.87	70.3/3.94-4.07
3,6	aDGalp	99.5/4.96	68.7/3.90	80.4/3.91	70.4/4.19	70.4/4.04	70.9/3.72-4.05
3	bDGlcp	105.8/4.56	74.2/3.32	76.9/3.52	70.2/3.61	75.5/3.61	66.0/3.69-4.00
2	Ac		23.7-24.1/2.03-2.06
	bDGalpN	104.1/4.71	52.7/4.06	81.4/3.89	69.1/4.15	75.8/3.69	62.2/3.77-3.80

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

892.811 g/mol (C₃₄H₅₆R₂N₂O₂₅, R = next and previous repeats, not counted in mol weight)