Table I. Proteomic analysis of the C. difficile exoproteome. C. difficile cells were grown in minimal medium until the beginning of the stationary phase. Conditioned medium was collected, filtered, concentrated, and analyzed with SDS-PAGE (supplemental Fig. S1A). Following in-gel digestion with trypsin, proteins were identified via LC–ion trap MS/MS and database searching (supplemental Tables S1 and S2). From this list, only proteins containing a signal peptide were selected and sorted based on the sequence coverage. Prediction of cell wall binding motifs, anchors, and subcellular localization was performed using NCBI Conserved Domain Search, the LipoP server, and PSORTB.

Locus	Protein name	Protein I.D.	SignalP cleavage	LPP lipid attachment	Cell wall binding motif	Localization (PSORTB)	Sequence coverage	Unique peptides
CD2793	Precursor of the S-layer proteins	Q183M8	Yes		CW binding 2	Cell wall	80	63
CD2768	Putative cell-wall hydrolase	Q183K6	Yes		SH3_3	Extracellular	61	11
CD2767	Putative cell surface protein	Q183K2	Yes		CW binding 2	Cell wall	48	28
CD2830	Uncharacterized protein	Q183R7	Yes			Unknown	47	10
CD0873	ABC-type transport system, sugar-family extracellular solute-binding protein	Q18A65	Yes	Yes		Unknown	45	11
CD2791	Cell wall binding protein	Q183M5	Yes		CW binding 2	Cell wall	44	25
CD1469	Putative cell surface protein putative penicillin-binding protein	Q18BY9	Yes		CW binding 2	Unknown	43	54
CD1522	Putative polysaccharide deacetylase	Q18C42	No			Extracellular	43	12
CD2784	Putative N-acetylmuramoyl-l-alanine amidase,autolysin (EC 3.5.1.28)	Q183L9	Yes		CW binding 2	Cell wall	42	29
CD2193	Fragment of putative cell wall-binding protein (N-terminal region) pseudogene	Q185Y1	Yes		CW binding 2	Cell wall	39	18
CD1156	Uncharacterized protein	Q18B20	No	Yes		Unknown	37	16
CD0183	Putative cell wall hydrolase	Q18CS3	Yes		SH3_3	Extracellular	36	10
CD2672	ABC-type transport system, oligopeptide-family solute-binding protein	Q183A6	Yes	Yes		Cell wall	34	17
CD1135	Putative SH3-domain protein	Q18AZ7	Yes		SH3_3	Cell wall	33	12
CD1035	Putative N-acetylmuramoyl-l-alanine amidase,autolysin (EC 3.5.1.28)	Q18AN5	Yes		CW binding 2	Cytoplasmic/membrane	31	19
CD2388	Putative peptidoglycan-binding/hydrolysing protein	Q181X4	Yes		PG_binding_1	Extracellular	31	8
CD2789	Cell surface protein	Q183M7	Yes		CW binding 2	Cell wall	30	17
CD2798	Cell wall binding protein	Q183N5	Yes		CW binding 2	Cell wall	29	10
CD2127	Putative exported protein	Q185R5	No			Cytoplasmic/membrane	29	6
CD2797	Putative calcium-binding adhesion protein	Q183N3	Yes		FA58C (discoin)	Unknown	28	51
CD1304	Acd (mannosyl-glycoprotein endo-β-N-acetylglucosamidase) (EC 3.2.1.96)	Q5S4P9	Yes		SH3_3	Extracellular	28	15
CD2408	Putative multidrug efflux pump	Q181Z3	Yes			Unknown	27	9
CD3043	Uncharacterized protein	Q184N0	No			Unknown	25	6
CD0844	Putative cell wall–binding protein	Q18A40	Yes		CW binding 2	Cell wall	25	8
CD2794	Cell wall binding protein	Q183N1	No		CW binding 2	Cell wall	24	14
CD2177	ABC-type transport system,cystine/aminoacid-family extracellular solute-binding protein	Q185W5	No	Yes		Unknown	23	4
CD2787	Cell surface protein (EC 3.4.22.15)	Q183M1	Yes		CW binding 2	Cell wall	22	12
CD2402	Putative cell wall hydrolase phosphatase-associated protein (EC 3.4.-.-)	Q181Y8	Yes		SH3_3	Extracellular	21	8
CD3669	Putative exported protein	Q181S4	Yes	Yes		Unknown	18	4
CD2795	Cell wall binding protein	Q183N0	Yes		CW binding 2	Cell wall	16	7
CD3192	Putative cell surface peptidase, M4 family	Q181V6	Yes		CW binding 2	Cell wall	15	6
CD2796	Cell surface protein	Q183N2	Yes		CW binding 2	Cell wall	10	3
CD2518	Cell surface protein	Q182K4	Yes		CW binding 2	Cell wall	9	3
CD3366	Putative exported protein	Q180L0	Yes			Unknown	9	2
CD0545	Uncharacterized protein	Q188Y7	Yes	Yes		Unknown	8	2
CD0514	Putative hemagglutinin/adhesin	Q188W0	Yes		CW binding 2	Cell wall	7	10
CD0855	ABC-type transport system, oligopeptide-family extracellular solute-binding protein	Q18A51	No	Yes		Cell wall	6	2
CD3145	Putative serine-aspartate repeat-containing protein SdrF	Q184Y2	Yes			Extracellular	3	2
CD2831	Putative adhesin	Q183R6	Yes			Cell wall	2	2