1. 220 160 120 100 90 80 70 60 50 40 30 15 min 30 min 45 min 60 min KDa Figure S1. SDS-PAGE of supernatant after incubation in digestion buffer. L. monocytogenes cells were incubated in digest buffer for 15, 30, 45 and 60 minutes and the supernatant analyzed by SDS-PAGE and silver staining. See Supporting Information SI.3 for details. Figure S1

2. Figure S2. Colony-forming unit (CFU) count of L. monocytogenes cells after incubation in digestion buffer. Cells (7 x 10 9 ) were incubated in digest buffer for 15, 30, 45 and 60 minutes. Cells were subsequently pelleted, resuspended in 1ml of 15% glycerol in Brain Heart Infusion (BHI) broth and plated at appropriate concentrations for CFU counting. For each time point, at least triplicate CFU counts were made. Error bars represent standard deviation. The x-axis shows incubation time (minutes) versus cell concentration. Cells numbers were reduced after incubation in digest buffer at 45 and 60 minutes. See Supporting Information SI.3 for details. Figure S2

3. Figure S3 Cell Concentration (x10 9 CFU) UntreatedTrypsin-Treated Figure S3. Colony-forming unit (CFU) count of trypsin treated and untreated L. monocytogenes cells. Cells (7 x 10 9 ) were taken from a culture grown to 0.6 O.D. 620nm and washed 3 times with PBS prior to incubation in 0.15ml of digest buffer. For the trypsin treated sample, 1.25 µg of trypsin was added for 30 min at 37 o C with 50 rpm agitation. Untreated sample was incubated for 30 min in digest buffer only. Cell pellets were recovered and re- suspended in 1ml of 15% glycerol in PBS and stored at -20 o C. Cell suspensions were thawed once for serial dilution and CFU count. The x-axis shows treatment groups: Untreated and Trypsin-Treated. Six independent experiments were performed for each condition. Mann-Whitney U test was performed to assess possible difference between untreated and trypsin-treated sample groups. Mann-Whitney U test statistic is 18 with a two-tailed probability of 1.0000. The two sample groups are not significantly different (P≥0.05, two-tailed test).

4. LMOf2365_0546 Negative Control Phase Contrast Figure S4a Figure S4a. Immunofluorescence microscopy of live L. monocytogenes cells using specific LMOf2365_0546 rabbit serum or pre-immunization serum as negative control. See Supporting Information SI.4 for details.

5. LMOf2365_2742 Negative Control Phase Contrast Figure S4b Figure S4b. Immunofluorescence microscopy of live L. monocytogenes cells using specific LMOf2365_2742 rabbit serum or pre-immunization serum as negative control. See Supporting Information SI.4 for details. Supplementary Table S1 (Separate as Excel File) Proteins of L. monocytogenes strain LI0521 (serotype 4b) identified in untreated and trypsin-treated cells. Supplementary Table S2 (Separate as Excel File) Proteins identifications observed in only one trial. Supplementary Table S3 (Separate as Excel File) Information on identified peptides.