Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

capsular polysaccharide, capsule degrading depolymerase, Klebsiella phage

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3	aDGalp	95.8	68.5	78.1	72.3	67.9	61.9
3	aDGalpA	101.5	67.9	75.0	67.9	72.8	176.2
	aLFucp	101.5	68.4	78.3	72.7	67.8	16.1


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3	aDGalp	5.25	4.09	4.04	3.92	4.20	3.74
3	aDGalpA	5.31	4.00	4.14	4.55	4.37	-
	aLFucp	5.21	3.96	4.06	3.90	4.18	1.18


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3	aDGalp	95.8/5.25	68.5/4.09	78.1/4.04	72.3/3.92	67.9/4.20	61.9/3.74
3	aDGalpA	101.5/5.31	67.9/4.00	75.0/4.14	67.9/4.55	72.8/4.37	
	aLFucp	101.5/5.21	68.4/3.96	78.3/4.06	72.7/3.90	67.8/4.18	16.1/1.18

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

484.407 g/mol (C₁₈H₂₈R₂O₁₅, R = next and previous repeats, not counted in mol weight)

Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

capsular polysaccharide, capsule degrading depolymerase, Klebsiella phage

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3,3,3	aDGalp	95.8	68.1	80.0	70.0	71.3	61.9
3,3,3,3	bDGalp	105.2	70.5	77.7	65.6	75.6	61.9
3,3,3	bDGalf	110.5	81.4	85.3	80.5	71.0	63.8
3,3,4	aDGalp	101.2	69.9	70.0	69.5	71.4	60.9
3,3	aDGalp	100.8	68.7	77.7	79.1	71.4	60.9
3	bDGalf
4	aDGalp
	aDGalp


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3,3,3	aDGalp	5.19	4.05	4.16	4.29	4.24	3.76
3,3,3,3	bDGalp	4.69	3.75	3.81	4.19	3.68	3.76
3,3,3	bDGalf	5.22	4.35	4.09	4.31	3.86	3.69
3,3,4	aDGalp	5.01	3.83	3.92	4.07	4.24	3.79
3,3	aDGalp	5.10	4.10	3.92	4.18	4.24	3.79
3	bDGalf
4	aDGalp
	aDGalp


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3,3,3	aDGalp	95.8/5.19	68.1/4.05	80.0/4.16	70.0/4.29	71.3/4.24	61.9/3.76
3,3,3,3	bDGalp	105.2/4.69	70.5/3.75	77.7/3.81	65.6/4.19	75.6/3.68	61.9/3.76
3,3,3	bDGalf	110.5/5.22	81.4/4.35	85.3/4.09	80.5/4.31	71.0/3.86	63.8/3.69
3,3,4	aDGalp	101.2/5.01	69.9/3.83	70.0/3.92	69.5/4.07	71.4/4.24	60.9/3.79
3,3	aDGalp	100.8/5.10	68.7/4.10	77.7/3.92	79.1/4.18	71.4/4.24	60.9/3.79
3	bDGalf
4	aDGalp
	aDGalp

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

Warning = sub-repeat ending at aDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at bDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at aDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at bDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at aDGalp: unknown unit count was assumed to be 3
Warning = sub-repeat ending at bDGalp: unknown unit count was assumed to be 3
OC[C@@H](O)[C@@H]1O[C@@H](O[C@H]2[C@@H](O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@@H](CO)O[C@H](O[C@H]3[C@H]([C@H](O)CO)O[C@@H](O[C@H]4[C@@H](O[C@H]5O[C@H](CO)[C@H](O)[C@H](O)[C@H]5O)[C@@H](CO)O[C@H](O[C@H]5[C@H]([C@H](O)CO)O[C@@H](O[C@H]6[C@@H](O[C@H]7O[C@H](CO)[C@H](O)[C@H](O)[C@H]7O)[C@@H](CO)O[C@H](O[C@H]7[C@H]([C@H](O)CO)O[C@@H](O[C@H]8[C@@H](O[C@H]9O[C@H](CO)[C@H](O)[C@H](O)[C@H]9O)[C@@H](CO)O[C@H](O)[C@@H]8O)[C@@H]7O)[C@@H]6O)[C@@H]5O)[C@@H]4O)[C@@H]3O)[C@@H]2O)[C@H](O)[C@H]1O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O[C@H]2O[C@H](CO)[C@H](O)[C@H](O[C@@H]3O[C@H](CO)[C@H](O)[C@H](O[C@H]4O[C@H](CO)[C@H](O)[C@H](O[C@@H]5O[C@H](CO)[C@H](O)[C@H](O[C@H]6O[C@H](CO)[C@H](O)[C@H](O)[C@H]6O)[C@H]5O)[C@H]4O)[C@H]3O)[C@H]2O)[C@H]1O

2936.553 g/mol (C₁₀₈H₁₈₂O₉₁)