Microscopic investigations

In this section, the morphology and membrane integrity of cells treated with endolysin and/or HHP were microscopically investigated to get a better understanding about the effect of endolysin on the physiological state and pressure stability of the cell.

3.2.2.1 LIVE/DEAD staining of screening experiments

Figure 21 shows a LIVE/DEAD staining of exponential-phase L. monocytogenes and S. aureus cells incubated with an increasing concentration of endolysin or lysostaphin. The experimental set-up was the same as used for the screening experiments in section 3.2.1.2.1 and 3.2.1.3.1. Since the results were similar for the different S. aureus and L. monocytogenes strains, only one single strain per species is shown. In Figure 21, it is clearly visible that the cell membrane of S. aureus was still intact after incubating with 0.03 µg/mL endolysin or lysostaphin (as indicated by green fluorescence). With higher concentrations, there is an increased loss of membrane integrity (as indicated by red fluorescence) which nicely corresponds to the inactivation data in Table 24.

A somewhat different trend was visible for L. monocytogenes cells incubated with endolysin. First, at a concentration of 0.16-0.8 µg/mL PlyP40 or PlyP825, the typical rod shaped cell morphology was lost and circular shaped cell bodies, postulated to be protoplasts, were formed. Interestingly, some of these circular cells showed no red fluorescence after LIVE/DEAD staining, indicating the formation of protoplasts with an intact cell membrane. Secondly, although the structure of every single cell was lost and showed red fluorescence after incubation with 4 µg/mL PlyP40 or PlyP825, circular shaped cells with no sign of a damaged membrane were again detected at even higher concentrations. This indicates that endolysin PlyP40/PlyP825 have a similar effect on Listeria cells, whether at a concentration of 0.8 or above 4 µg/mL, a phenomenon also observed in the inactivation data of the screening experiment (Table 22) and referred to as Eagle effect. For endolysin Ply511, a concentration of 0.8 and 4 µg/mL also led to the formation of circular shaped cells of which some also showed green fluorescence. Though, in contrast to endolysin PlyP40 and PlyP825, all cells had a compromised cell membrane at an even higher PlyP511 concentration (which is also in accordance with the increased inactivation at higher Ply511 concentrations as observed in the screening experiment).

60 RESULTS

3.2.2.2 Endolysin-induced formation of L. monocytogenes protoplasts

To examine the loss of cellular shape more closely (as seen for Listeria in the previous section), cells were microscopically observed while endolysin PlyP40 was added directly from the stock solution to the microscopic slide. As becomes clear from Figure 22, increasingly more cells lost their rod shape and formed circular shaped cell bodies over time. After 60 min of coincubation with a high concentration of PlyP40, most Listeria cells had lost their rod shape. After even longer incubations time, only circular shaped cell bodies were left.

Figure 21: LIVE/DEAD staining of L. monocytogenes and S. aureus cells incubated with endolysin. Exponential-phase L. monocytogenes (TMW 2.599) or S. aureus (TMW 2.424) cells at an inoculum of ca. 10⁷ cells/mL were incubated with IPB (control) or different concentrations of endolysin (0.3, 0.16, 0.8, 4, 20, or 100 µg/mL). After ca. 75 min of coincubation with

endolysin, cells were stained with PI (red fluorescence) and SYTO 9 (green fluorescence), captured under a microscopic slide, and analysed with epifluorescent microscopy. Images of the same microscopic field were merged and the contrast and brightness were adjusted for optimal representation. A representative part of the images was cut out and enlarged for

better visiblity. Scale bar = 10 µm.

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In order to get more insights on the physiological state of the circular shaped cell bodies or protoplasts, their membrane integrity was examined by LIVE/DEAD staining. Cells were therefore mixed with the nucleic acid stains PI and SYTO 9 before being captured between the microscopic slide and the addition of endolysin. Figure 23 shows that coincubation of L. monocytogenes with endolysin Ply511 or PlyP825 for 60 minutes led to the formation of protoplasts of which most had a compromised cell membrane (as indicated by red fluorescence). Though, for some protoplasts, there was no indication of a damaged membrane (pointed out by the white arrows). These results thereby indicate that although both Ply511 and Ply825 cause Listeria to lose their rod-shape as a result of peptidoglycan breakdown, their action can result in circular shaped cells with an intact membrane (as could be examined by LIVE/DEAD staining).

Figure 22: Time-lapse phase contrast microscopic images of L. monocytogenes cells incubated with PlyP40. Exponential-phase L. monocytogenes cells (strain TMW 2.599 at an inoculum of ca. 10⁹ cells/mL IPB) were captured under a microscopic

slide and PlyP40 was added directly from the stock (1 mg/mL). Microscopic images of the same microscopic field were taken every minute (only t1, t30, t60 min are provided for illustrative purposes).

Scale bar = 5 µm

Figure 23: Membrane integrity of L. monocytogenes protoplast after incubation with Ply511 or PlyP825. Exponential-phase L. monocytogenes cells (strain TMW 2.599 at an inoculum of ca. 10⁹ cells/mL IPB) were stained with PI (red fluorescence) and SYTO 9 (green fluorescence), captured under a microscopic slide, and either Ply511 (top images) or

PlyP825 (bottom images) was added directly from the stock. Phase contrast and epifluorescent images of the same microscopic field were taken after 60 min of coincubation with endolysin. The contrast and brightness of epifluorescent microscopic images was adjusted for optimal representation. White arrows indicate circulate shaped cells for which no PI

fluorescence was visible. Scale bar = 5 µm.

Plyp825Ply511

62 RESULTS

3.2.2.3 Pressure-stability of L. monocytogenes protoplasts

The effect of HHP on the membrane integrity of fresh L. monocytogenes cells or those incubated with a very high concentration of endolysin PlyP825 (500 µg/mL) was examined by LIVE/DEAD staining. The cells were stained after HHP treatment and only a more permanent loss of membrane integrity was therefore assessed (in contrast to the transient permeabilization of the membrane during pressure treatment). Figure 24 shows that the membrane of L. monocytogenes cells was stable up to a pressure level of 300 MPa (30 °C, 1 min). At higher pressure levels, the number of cells with an impaired membrane steadily increased. This is perfectly in line with the preliminary experiments described in section 3.2.1.1, where 300 MPa was found to be the maximum pressure level which did not cause major inactivation. When cells were first incubated with 500 µg/mL PlyP825 for 2 hours, mostly circular shaped cells were visible of which about half did not show red fluorescent staining (indicating an intact membrane). A pressure level of 100 MPa (30 °C, 1 min) did not change this observation. At pressure levels of 200 MPa or higher, no more circular shaped cells with an intact cellular membrane could be detected. This indicates that the membrane of these cells is impaired and more sensitive to HHP than cells without endolysin treatment.