In skin, Cutibacterium acnes (former Propionibacterium acnes) can behave as an opportunistic pathogen, depending on the strain and environmental conditions. Acneic strains of C. acnes form biofilms inside skin-gland hollows, inducing inflammation and skin disorders. The essential exogenous products of C. acnes accumulate in the extracellular matrix of the biofilm, conferring essential bacterial functions to this structure. However, little is known about the actual composition of the biofilm matrix of C. acnes. Here, we developed a new technique for the extraction of the biofilm matrix of Gram-positive bacteria without the use of chemical or enzymatic digestion, known to be a source of artifacts. Our method is based on the physical separation of the cells and matrix of sonicated biofilms by ultracentrifugation through a CsCl gradient. Biofilms were grown on the surface of cellulose acetate filters, and the biomass was collected without contamination by the growth medium. The biofilm matrix of the acneic C. acnes RT5 strain appears to consist mainly of polysaccharides. The following is the ratio of the main matrix components: 62.6% polysaccharides, 9.6% proteins, 4.0% DNA, and 23.8% other compounds (porphyrins precursors and other). The chemical structure of the major polysaccharide was determined using a nuclear magnetic resonance technique, the formula being →6)-α-D-Galp-(1→4)-β-D-ManpNAc3NAcA-(1→6)-α-D-Glcp-(1→4)-β-D-ManpNAc3NAcA-(1→3)-β-GalpNAc-(1→. We detected 447 proteins in the matrix, of which the most abundant were the chaperonin GroL, the elongation factors EF-Tu and EF-G, several enzymes of glycolysis, and proteins of unknown function. The matrix also contained more than 20 hydrolases of various substrata, pathogenicity factors, and many intracellular proteins and enzymes. We also performed surface-enhanced Raman spectroscopy analysis of the C. acnes RT5 matrix for the first time, providing the surface-enhanced Raman scattering (SERS) profiles of the C. acnes RT5 biofilm matrix and biofilm biomass. The difference between the matrix and biofilm biomass spectra showed successful matrix extraction rather than simply the presence of cell debris after sonication. These data show the complexity of the biofilm matrix composition and should be essential for the development of new anti-C. acnes biofilms and potential antibiofilm drugs.

polysaccharides, nuclear magnetic resonance, proteins, biofilms, biofilm matrix, Cutibacterium acnes, Orbitrap mass spectrometry, surface-enhanced Raman spectroscopy

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,6,4	aDGalp	99.8	69.5	70.4	70.0	71.5	68.1
3,4,6,2	Ac	175.4	23.4
3,4,6,3	Ac	175.4	23.4
3,4,6	bDManpN3NA	101.3	52.9	54.5	79.7	72.9	175.9
3,4	aDGlcp	99.8	72.7	74.1	70.4	72.3	69.3
3,2	Ac	175.8	23.4
3,3	Ac	175.8	23.4
3	bDManpN3NA	101.1	52.6	54.3	79.6	73.0	176.0
2	Ac	176.4	23.4
	bDGalpN	102.7	52.0	82.3	68.2	76.2	62.5


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,6,4	aDGalp	5.14	3.69	3.74	3.91	3.95	3.73-3.89
3,4,6,2	Ac	-	1.97
3,4,6,3	Ac	-	1.97
3,4,6	bDManpN3NA	4.99	4.30	4.29	3.94	3.88	-
3,4	aDGlcp	5.08	3.35	3.59	3.41	3.73	3.88-3.98
3,2	Ac	-	2.01
3,3	Ac	-	2.01
3	bDManpN3NA	4.89	4.41	4.32	3.96	3.90	-
2	Ac	-	2.07
	bDGalpN	4.64	3.97	3.85	4.14	3.73	3.76


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,6,4	aDGalp	99.8/5.14	69.5/3.69	70.4/3.74	70.0/3.91	71.5/3.95	68.1/3.73-3.89
3,4,6,2	Ac		23.4/1.97
3,4,6,3	Ac		23.4/1.97
3,4,6	bDManpN3NA	101.3/4.99	52.9/4.30	54.5/4.29	79.7/3.94	72.9/3.88	
3,4	aDGlcp	99.8/5.08	72.7/3.35	74.1/3.59	70.4/3.41	72.3/3.73	69.3/3.88-3.98
3,2	Ac		23.4/2.01
3,3	Ac		23.4/2.01
3	bDManpN3NA	101.1/4.89	52.6/4.41	54.3/4.32	79.6/3.96	73.0/3.90	
2	Ac		23.4/2.07
	bDGalpN	102.7/4.64	52.0/3.97	82.3/3.85	68.2/4.14	76.2/3.73	62.5/3.76

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]OC[C@H]1O[C@H](O[C@@H]2[C@@H](C(=O)O)O[C@@H](OC[C@H]3O[C@H](O[C@@H]4[C@@H](C(=O)O)O[C@@H](O[C@@H]5[C@@H](NC(C)=O)[C@H]([*])O[C@H](CO)[C@@H]5O)[C@@H](NC(C)=O)[C@H]4NC(C)=O)[C@H](O)[C@@H](O)[C@@H]3O)[C@@H](NC(C)=O)[C@H]2NC(C)=O)[C@H](O)[C@@H](O)[C@H]1O

1043.936 g/mol (C₄₀H₆₁R₂N₅O₂₇, R = next and previous repeats, not counted in mol weight)