Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organisation refers it to its <> category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-depolymerasing enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89 and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae The linear double-stranded DNA phage genomes of 41,105-42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K-units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that cleave specifically the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K-units.IMPORTANCE Acinetobacter baumannii, a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K-types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidate to the cocktail designed to control A. baumannii with different K-types

structure, Acinetobacter baumannii, capsular polysaccharide, bacteriophage, glycosidase, capsular type, tailspike depolymerase

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
4,3,3,2	Ac	174.3-175.7	23.3-23.7
4,3,3	aDGlcpN	95.1	54.7	71.0	72.1	73.1	61.5
4,3,6	bDGalp	104.7	72.0	74.0	69.9	76.4	62.2
4,3	bDGalp	105.9	70.3	78.0	66.0	74.5	70.3
4,2	Ac	174.4-176.1	22.9-23.7
4	bDGalpN	103.2	52.6	81.8	69.2	75.9	62.3
5	Ac	174.3-175.7	23.3-23.7
	bXLegp	?	?	40.9	78.0	52.1	70.9	54.4	67.7	20.4
7	Ac	174.3-175.7	23.3-23.7
7	lS3HOBut	?	46.1	66.3-66.4	23.5-23.7


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
4,3,3,2	Ac	-	1.95-2.04
4,3,3	aDGlcpN	5.03	3.96	3.53	3.82	3.97	3.73-3.80
4,3,6	bDGalp	4.41	3.50	3.63	3.91	3.68	3.75-3.75
4,3	bDGalp	4.47	3.60	3.69	4.11	3.78	3.86-4.01
4,2	Ac	-	1.95-2.04
4	bDGalpN	4.57	3.91	3.84	4.19	3.67	3.81-3.81
5	Ac	-	1.95-2.04
	bXLegp	-	-	1.88-2.42	4.00	3.80	4.23	3.83	3.84	1.13
7	Ac	-	1.95-2.04
7	lS3HOBut	-	2.36-2.40	4.16-4.18	1.23-1.24


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
4,3,3,2	Ac		23.3-23.7/1.95-2.04
4,3,3	aDGlcpN	95.1/5.03	54.7/3.96	71.0/3.53	72.1/3.82	73.1/3.97	61.5/3.73-3.80
4,3,6	bDGalp	104.7/4.41	72.0/3.50	74.0/3.63	69.9/3.91	76.4/3.68	62.2/3.75-3.75
4,3	bDGalp	105.9/4.47	70.3/3.60	78.0/3.69	66.0/4.11	74.5/3.78	70.3/3.86-4.01
4,2	Ac		22.9-23.7/1.95-2.04
4	bDGalpN	103.2/4.57	52.6/3.91	81.8/3.84	69.2/4.19	75.9/3.67	62.3/3.81-3.81
5	Ac		23.3-23.7/1.95-2.04
	bXLegp			40.9/1.88-2.42	78.0/4.00	52.1/3.80	70.9/4.23	54.4/3.83	67.7/3.84	20.4/1.13
7	Ac		23.3-23.7/1.95-2.04
7	lS3HOBut		46.1/2.36-2.40	66.3-66.4/4.16-4.18	23.5-23.7/1.23-1.24

The spectrum also has 3 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@@H]1[C@@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O[C@@H]3O[C@H](CO[C@@H]4O[C@H](CO)[C@H](O)[C@H](O)[C@H]4O)[C@H](O)[C@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4NC(C)=O)[C@H]3O)[C@H]2NC(C)=O)C[C@@](O)(C(=O)O)O[C@H]1[C@H](NC(C)=O)[C@@H](C)O

1064.995 g/mol (C₄₁H₆₈N₄O₂₈)